Born in Caroline County, Virginia, Clark moved with his family to Louisville, Kentucky in 1785. After his brother George joined the army, William Clark followed, and participated in several local militia campaigns. He was commissioned a lieutenant in the regular army in 1792, and was assigned to Anthony Wayne's Legion of the United States, where he served a four-year tour and participated in the Battle of Fallen Timbers. Also during this period, one of the men briefly under his command was Meriwether Lewis.

Clark left the army in 1796, spending time at his estate in Louisville and traveling from time to time. In 1803 he was asked by Lewis to share command of the newly-formed Corps of Discovery. Clark spent three years on the expedition, and although technically subordinate to Lewis in rank, exercised equal authority at Lewis's insistence. He concentrated chiefly on the drawing of maps, the management of the expedition's supplies, and the identification of native flora and fauna, and after returning in 1806 spent a great deal of time consolidating the information collected.

Clark was appointed a brigadier general of the militia and made superintendent of Indian affairs in the Louisiana Territory in 1807. He set up his headquarters for this in St. Louis, Missouri. When the Missouri Territory was formed in 1813 Clark was appointed governor. During the War of 1812 he led several campaigns, and established the first post in what is now Wisconsin.

After the war Clark returned to the administration of Indian affairs, employing various diplomatic and military measures in response to several uprisings in the area, such as the Black Hawk War. He also worked as a surveyor. His years as superintendent for Indian Affairs were very important. Clark's decisions on a daily basis as Indian Superintendent had an impact on individual lives far greater than his explorations. His region of influence in the 1830's was immense.

Clark married Julia Hancock on January 5, 1808 and had five children with her: Meriwether Lewis Clark named after his good friend Meriwether Lewis (1809-1881), William Preston Clark (1811-1840), Mary Margaret Clark (1814-1821), George Rogers Hancock Clark (1816-1858), and John Julius Clark (1818-1831). After Julia's death in 1820 he married her first cousin Harriet Kennerly Radford and had three children with her: Jefferson Kearny Clark (1824-1900), Edmund Clark (1826-1827) and Harriet Clark (dates unknown died as child). His second wife Harriet died in 1831.

Clark died in St. Louis and was buried in Bellefontaine Cemetery, where a 35-foot gray granite obelisk was erected to mark his grave. Although his family had established endowments to maintain the site, by the late 20th century the grave site had fallen into disrepair. His descendants raised $100,000 to rehabilitate the obelisk, and celebrated the rededication with a ceremony May 21, 2004, on the bicentennial of the start of his famous expedition. The ceremony was attended by the largest gathering of his descendants, re-enactors in period dress, and leaders from the Osage Nation, and the Lemhi band of the Shoshone Native American peoples.

The western American plant genus Clarkia (in the Evening primrose family Onagraceae), is named after him, as is the Western cutthroat trout (Oncorhynchus clarki). Several states have named a county in his honor: Arkansas, Idaho, Missouri, Montana, and Washington. He also has a star on the St. Louis Walk of Fame.