During his time in Georgia, Lewis enhanced his skills as a hunter and outdoorsman. He would often venture out in the middle of the night in the dead of winter with only his dogs to go hunting. Even at his early age he was interested in natural history, which would develop into a lifelong passion. His mother taught him how to gather wild herbs for medicinal purposes. It was also in the Broad River Valley that Lewis first dealt with a native Indian group. The Cherokee lived in antagonistic proximity to the white settlers, but Lewis seems to have been a champion for them amongst his own people. It was in Georgia that he met Eric Parker, who was the first to introduce him to the idea of traveling. At thirteen, he was sent back to Virginia for education by private tutors. One of these was Parson Matthew Maury, an uncle of Matthew Fontaine Maury. Parson Maury was a son of Charles Goodyear Maury who was Thomas Jefferson's teacher for two years. In 1793, Lewis graduated from Liberty Hall (now Washington and Lee University), joined the Virginia militia, and in 1794 he was sent as part of a detachment involved in putting down the Whiskey Rebellion.

In 1795 he joined the U.S. Army, as a Lieutenant, where he served until 1801, at one point in the detachment of William Clark, who would later become his companion in the Corps of Discovery.

On April 1, 1801, he was appointed as an aide by President Thomas Jefferson, whom he knew personally through Virginia society in Albemarle County. Lewis resided in the presidential mansion, and frequently conversed with various prominent figures in politics, the arts and other circles. Originally, he was to provide information on the politics of the United States Army, which had seen an influx of Federalist officers as a result of John Adams's "midnight appointments". When Jefferson began to formulate and to plan for an expedition across the continent, he chose Lewis to lead the expedition.

After returning from the expedition, Lewis received a reward of 1,600 acres (6.5 km²) of land. He also initially made arrangements to publish the Corp of Discovery journals but had difficulty completing his writing of the journals. In 1807, Jefferson appointed him governor of the Louisiana Territory; he settled in St. Louis. He died in 1809 on his journey to deliver his journals to a Washington publisher.

Lewis' record as an administrator is mixed. He established roads and was a strong proponent of the fur trade. His position was to protect the western lands from encroachment, which was not favorable to the rush of settlers looking to open new lands for settlements. But due to his quarreling with local political leaders, controversy over his approvals of trading licenses, land grant politics, Indian depredations, excessive drinking and a slow moving mail system, it appeared that Lewis was a poor administrator. He did not adequately keep in touch with his superiors in Washington.

Lewis was a Freemason, initiated, passed and raised in Door To Virtue Lodge No. 44 in Albemarle, Virginia, between 1796 and 1797. On August 2, 1808, Lewis and several of his acquaintances submitted a petition to the Grand Lodge of Pennsylvania in which they requested a dispensation to establish a lodge in St. Louis. Lewis was nominated and recommended to serve as the first Master of the proposed Lodge, which was warranted as Lodge No. 111 on September 16, 1808.

While modern historians generally accept his death as a suicide, there is some debate. Mrs. Grinder, the tavern keeper's wife, claimed Lewis acted strangely the night before his death. She said that during dinner Lewis stood and paced about the room talking to himself in the way one would speak to a lawyer. She observed his face to flush as if it had come on him in a fit. After he retired for the evening, Mrs. Grinder continued to hear him talking to himself. At some point in the night she heard multiple gunshots, and what she believed was someone asking for help. She claimed to be able to see Lewis through the slit in the door crawling back to his room. She never explained why, at the time, she didn't investigate further concerning Lewis's condition or the source of the gunshots. The next morning, she sent for Lewis's servants. They found him wounded and bloody, with part of his skull gone, but he lived for several hours. Mrs. Grinder's testimony is held as a point of contention from both sides of the murder - suicide debate. The murder advocates point to five conflicting testimonies as evidence that her testimony is fabricated, and the suicide advocates point to her testimony as proof of suicide.

When Clark and Jefferson were informed of Lewis' death, both accepted the conclusion of suicide. His mother and relatives contended it was murder. In later years a court of inquiry explored whether they could charge the husband of the tavern - keeper with Lewis' death. They dropped the inquiry for lack of evidence or motive.

From 1993 – 2010, many of Lewis' kin (through his sister Jane, as he had no children) sought to have the body exhumed for forensic analysis, to try to determine whether the death was a suicide. A Tennessee coroner's jury in 1996 recommended exhumation, but since Lewis is buried in a national park, the National Park Service must approve; they refused the request in 1998, citing possible disturbance to the bodies of more than 100 pioneers buried nearby. In 2008 the Department of Interior approved the exhumation, but that decision was rescinded in 2010 upon policy review, and the Department has stated the last decision is final. It is making improvements to the grave site and visitor facility.

The explorer was buried near present day Hohenwald, Tennessee, near his place of death. The State of Tennessee erected a monument over his grave in 1848. The Tennessee State Commission charged with locating the grave and erecting the monument wrote in its official report that it was likely Lewis was murdered. Today, the grave site is maintained by the Natchez Trace Parkway.

In 2009, the Lewis and Clark Trail Heritage Foundation organized a commemoration for Lewis in conjunction with their 41st annual meeting October 3–7, 2009. It included the first national memorial service at his gravesite. On October 7, 2009, the 200th anniversary of Lewis' death, about 2,500 people (Park Service estimate) from more than twenty - five states gathered at his grave. His life and achievements were acknowledged. Speakers included Clark descendant Peyton "Bud" Clark, Lewis collateral descendants Howell Bowen and Tom McSwain, and Stephanie Ambrose Tubbs (the daughter of the author Stephen Ambrose, who wrote Undaunted Courage, an award winning book about the Lewis and Clark expedition). A bronze bust of Lewis was dedicated to the Natchez Trace Parkway for a planned visitor center at the gravesite area. The District of Columbia and governors of twenty states sent flags flown over state capital buildings to be carried to Lewis' grave by residents of the states associated with the Lewis and Clark Trail.

This was the final bicentennial event honoring the Lewis and Clark Expedition. Re-enactors from the Lewis and Clark Bicentennial participated, and official attendees included Native American tribal chiefs and representatives from Jefferson's Monticello. The Lewis and Clark descendants, along with representatives of St. Louis Lodge #1, past presidents of the Lewis and Clark Trail Heritage Foundation, and the Daughters of the American Revolution, carried wreaths and led a formal procession to Lewis' grave. Samples of plants which Lewis discovered on the expedition were brought from the Trail states and laid on his grave. The U.S. Army was represented by the 101st Airborne Infantry Band and its Army chaplain.

For many years, Lewis' legacy was overlooked, inaccurately assessed, and somewhat tarnished by his alleged suicide. Yet his contributions to science, the exploration of the Western U.S., and the lore of great world explorers, are considered incalculable.

Four years after Lewis' death, Thomas Jefferson wrote:

Of courage undaunted, possessing a firmness and perseverance of purpose which nothing but impossibilities could divert from its direction, ... honest, disinterested, liberal, of sound understanding and a fidelity to truth so scrupulous that whatever he should report would be as certain as if seen by ourselves, with all these qualifications as if selected and implanted by nature in one body for this express purpose, I could have no hesitation in confiding the enterprise to him.

Jefferson also stated that Lewis had a "luminous and discriminating intellect."

The alpine plant Lewisia (family Portulacaceae), popular in rock gardens, is named after Lewis, as is Lewis's Woodpecker. A subspecies of cutthroat trout, the westslope cutthroat (Oncorhynchus clarki lewisi), is also named after him. Geographic names that honor him include Lewis County, Idaho, Lewis County, Kentucky; Lewis County, Tennessee; Lewisburg, Tennessee; Lewiston, Idaho; Lewis County, Washington; the U.S. Army fort Fort Lewis, Washington, the home of the US Army 1st Corps (I Corps), and especially Lewis and Clark County, Montana, the home of the capital city, Helena; the Lewis Range of Montana's Glacier National Park; Lewis Avenue in Phoenix, Arizona. A day use campground at Gates of the Mountains Wilderness, north of Helena, Meriwether Picnic site. A cave, Lewis and Clark Caverns between Three Forks and Whitehall, Montana. The US Navy Polaris nuclear submarine USS Lewis and Clark was named for him and William Clark.