The Ohio River is a 981-mile (1,579 km) long river in the United States. It is located at the boundary of the Midwestern and Southern United States, flowing in a southwesterly direction from western Pennsylvania to its mouth on the Mississippi River at the southern tip of Illinois. It is the third largest river by discharge volume in the United States and the largest tributary by volume of the north-south flowing Mississippi River that divides the eastern from western United States. It is also the 6th oldest river on the North American continent. The river flows through or along the border of six states, and its drainage basin includes parts of 14 states. Through its largest tributary, the Tennessee River, the basin includes several states of the southeastern U.S. It is the source of drinking water for five million people.

The lower Ohio River just below Louisville is obstructed by rapids known as the Falls of the Ohio where the elevation falls 26 feet (7.9 m) in 2 miles (3.2 km) restricting larger commercial navigation, although in the 18th and early 19th century its three deepest channels could be traversed by a wide variety of craft then in use. In 1830, the Louisville and Portland Canal (now the McAlpine Locks and Dam) bypassed the rapids, allowing even larger commercial and modern navigation from the Forks of the Ohio at Pittsburgh to the Port of New Orleans at the mouth of the Mississippi on the Gulf of Mexico.

The name "Ohio" comes from the Seneca, Ohi:yo', lit. "Good River". In his Notes on the State of Virginia published in 1781–82, Thomas Jefferson stated: "The Ohio is the most beautiful river on earth. Its current gentle, waters clear, and bosom smooth and unbroken by rocks and rapids, a single instance only excepted" (though Jefferson himself had not seen the Ohio) In the late 18th century, the river was the southern boundary of the Northwest Territory. It became a primary transportation route for pioneers during the westward expansion of the early U.S. Today, the Ohio River is one of the most polluted rivers in the United States.

The river is sometimes considered as the western extension of the Mason–Dixon Line that divided Pennsylvania from Maryland, and thus part of the border between free and slave territory, and between the Northern and Southern United States or Upper South. Where the river was narrow, it was crossed by thousands of slaves escaping to the North for freedom; many were helped by free blacks and whites of the Underground Railroad resistance movement.

The Ohio River is a climatic transition area, as its water runs along the periphery of the humid subtropical and humid continental climate areas. It is inhabited by fauna and flora of both climates. In winter, it regularly freezes over at Pittsburgh but rarely farther south toward Cincinnati and Louisville. Further down the river in places like Paducah and Owensboro, Kentucky, closer to its confluence with the Mississippi, the Ohio is ice-free year-round.