During the American Revolution, Rockbridge County was formed from a portion of Augusta County. The county seat, Lexington, was named after its counterpart in Massachusetts and established in 1777. Lexington Presbyterians attended Hall's Meeting House, five miles to the west of town, established in 1746. On 15 April 1789, the Lexington Presbyterian Church was authorized by Lexington Presbytery, as a new church development project of New Monmouth Presbyterian Church (formerly Hall's Meeting House).

The first minister in 1789 was the Rev. William Graham, who was also Rector of Liberty Hall Academy (now Washington & Lee University). At first the congregation met out of doors during the warmer months, either in an oak grove or under a tent, and in the county court house in the colder months. By 1799 the Lexington congregation began meeting in a new brick church located in what is today the NW corner of the Oak Grove Cemetery. For the first thirty years of its life, the Lexington Presbyterian Church, though meeting for worship separately, remained linked to New Monmouth Church, both churches sharing the same Session and pastor.

In the early 1840s, desiring a more comfortable place of worship in a more central location in town, the Female Working Society of the Presbyterian Church purchased and gave to the church the lot at the corner of Main and Nelson Streets on which the current building stands. The Greek Revival structure, designed by Thomas U. Walter, was completed in 1845. In 1895 the church added wings to accommodate continuing growth and activity. The building was enlarged, improved, and refurbished again in 1899. In 1979, the building was registered by the Virginia Historic Landmarks Commission as a Virginia Historic Landmark.

On July 18, 2000, the sanctuary burned, but it was reconstructed so that the exterior and much of the interior plan was retained. The congregation reoccupied the restored sanctuary on January 5, 2003. An interview with pastor Bill Klein and clerk-of-the-works Frank Parsons describing the recovery from the fire may be found on YouTube. This video was produced by New Holland Church Furniture.

A much-used lecture room stood beside the church building after 1845, sandwiched between the sanctuary and a private dwelling. In 1905 the dwelling was purchased from Mrs. Lucy Houghawout. A new classroom building had been erected in its place by 1907 and was enlarged in 1922. In 1952 the church purchased the land behind this classroom building through to Randolph Street, and by 1956 a new enlarged Christian Education Building had been completed. In May 1957, the new building was named Murray Hall for Dr. Joseph James Murray, pastor of thirty-three years. In 2012 Murray Hall was extensively renovated for handicapped accessibility.

The old Presbyterian manse, located at 6 White Street, completed in 1848, was fashioned from bricks salvaged when the original church building was razed. It was in continuous use to house the Lexington Presbyterian Church pastor and his family until it was sold in October of 2016. Click on the link for more information about this historic building, which was very avant-garde for its day, and its preservation.

General Thomas J. ("Stonewall") Jackson was a member of this church from the year of his arrival in 1851 as a professor at the Virginia Military Institute until his death in 1863. Until his departure from Lexington in April of 1861, Jackson taught a Sunday School Class that was well-attended by the town's African-American population. Jackson's next return to Lexington was for burial in the Presbyterian Cemetery on 15 May 1863.

The Lexington Presbyterian Church has had in its history seventeen pastors and six stated supply ministers. From its membership have come thirty-six pastors, and some fourteen have gone to the foreign mission field from this church.