Presbyterian Scotch-Irish immigrants made up a large portion of the earliest settlers of European stock in what is now West Virginia, arriving in the back country in the early 18th century. They entered through the present Eastern Panhandle; through the northwest, by coming down the Ohio from Pittsburgh; and through the southeast, from the Valley of Virginia into Tygart Valley and portions of Monroe, Greenbrier, Kanawha, and Pocahontas counties.

Dispatched by the Synod of Philadelphia in 1720, Daniel McGill organized Presbyterians into a "church order" at "Potomoke," south of the Potomac River, perhaps at or near Shepherdstown. Other early Presbyterian churches were at Bullskin, Jefferson County, 1736; Tuscarora, Berkeley County, 1740; Back Creek, Hedgesville, 1741; Moorefield, 1741; and Falling Waters, Berkeley County, 1745. Other pre-Revolutionary churches were planted in Hampshire, Mineral, Jefferson, and Hardy counties, 1761-73. In the Revolutionary Era, churches took root in Huttonsville, Pickaway, Romney, Gerrardstown, Lewisburg, Union, Renick, Wheeling, Charles Town, Morgantown, West Liberty, Weirton Heights, Pughtown, Hillsboro, and Follansbee, 1781-98.

In 1789, the initial meeting of the Presbyterian General Assembly adopted a strategic policy to send missionaries to the American frontier settlements in order to form new congregations. It was the first denomination to regularly send missionaries to the West, according to church historian Ernest Trice Thompson. Many missionaries were unmarried young graduates of Hampden-Sydney College or Liberty Hall Academy, the precursor of Washington and Lee University.

John McCue was among the Presbyterian missionaries who followed the early settlers into the region west of the Alleghenies. McCue planted churches in Lewisburg, Union, and Spring Creek (Renick) in 1783, the historic "three cornerstones" which were indispensable to the future development of Presbyterianism westward to the Ohio River.

In 1808, the Committee on Missions of the Synod of Virginia sent John McElhenney as a Presbyterian evangelist in Greenbrier and Monroe counties. He was installed as pastor of churches in Lewisburg and Union the following summer. McElhenney served as pastor of the Union Presbyterian Church until 1834. He served the Old Stone Presbyterian Church of Lewisburg until his death in 1871. Soon after their arrival, John and Rebecca McElhenney founded the Lewisburg Academy, where John was principal and instructor for two decades. McElhenney and those trained by him organized congregations in the Greenbrier Valley, in Charleston, and in the Ohio Valley at Point Pleasant and Parkersburg.

Among the graduates of Lewisburg Academy and Washington College (now Washington and Lee) was Henry Ruffner, the son of Col. David Ruffner, a founder of the salt industry in the Kanawha Valley. Henry Ruffner became the first principal of Charleston's Mercer Academy. In 1819, he founded the Kanawha Church of Charleston and the Kanawha Salines Church at present Malden. Ruffner was appointed professor at Washington College in 1819 and served as president from 1836 to 1848. In 1841, he gathered with other western Virginians at a convention in Clarksburg and called for a system of free public schools. Ruffner's vision was carried forward in the next generation by his son, William Henry Ruffner, and significantly influenced the public education movements in Virginia and West Virginia.

Henry Ruffner was an opponent of slavery, an institution that helped split the Presbyterian denomination into northern and southern factions as the Civil War commenced. By the time of the creation of West Virginia in 1863, several Presbyterian congregations were divided. Some favored membership in the Northern Presbyterian denomination; others favored alignment with the Southern General Assembly. Charleston's Presbyterian congregation remained neutral during the war, but in 1872 split into the First Presbyterian Church (Southern) and the Kanawha Presbyterian Church (Northern).

With the birth of the First Presbyterian Church of Williamson in 1894, the denomination began a concerted movement into the developing coalfields south of Charleston. Southern West Virginia coal camps presented a fertile and challenging home mission. While many new ministers were called to the coalfields, city and town ministers also left their local parishes after Sunday morning services to teach and preach in coal communities along the railroads in the afternoon and the evening.

After World War II, the Presbyterian Church in West Virginia experienced dynamic growth, especially in Kanawha Presbytery under the leadership of Frank McCutchan Ryburn, who served as Evangelist and Superintendent of Home Missions, 1948-64. By 1950, Charleston and surrounding communities held one of the largest concentrations of Presbyterians, in proportion to population, in the country.

Despite losses in membership in West Virginia in recent decades, Presbyterians and their institutions have contributed greatly to the state's moral and social fabric. Many Presbyterians have served West Virginia as governor and in the U.S. Senate and House of Representatives. During and beyond the Civil Rights era of the 1950s and 1960s prophetic Presbyterians with West Virginia ties, such as Angie King, Robert B. McNeill, Dunbar H. Ogden, and William A. Benfield Jr., were courageous voices for racial justice and harmony.

Women have had a major impact on West Virginia Presbyterianism through historical societies, auxiliaries, and organizations which have stressed Bible study, prayer, global mission, hunger relief, peacemaking, and advocacy for justice and mercy. Since the reunion of the major northern and southern streams of American Presbyterianism in 1983, as the Presbyterian Church (U.S.A.), West Virginia women have occupied significant leadership positions. From 1985 to 1998, Dorothy I. MacConkey was president of Davis & Elkins College, a Presbyterian-related institution founded in 1904. Patricia L. Kennedy of the Montgomery Presbyterian Church served as chair of the General Assembly Council, Presbyterian Church (U.S.A.), 1990-91.

There were a total of 23,650 members of 202 “Presbyterian U.S.A.” churches in West Virginia, according to a 2000 church survey. By 2022, there were 6,191 members in 120 churches in the Presbytery of West Virginia; however, that number does not include approximately 50 to 60 churches in the Eastern and Northern Panhandles or about 10 “Presbyterian Church of America” churches.