Jefferson County is the easternmost county in the U.S. state of West Virginia. It is located in the Shenandoah Valley in the eastern panhandle of West Virginia. As of the 2020 census, the population was 57,701. Its county seat is Charles Town. The county was founded in 1801, and today is part of the Washington metropolitan area.

Jefferson County was established on October 26, 1801 from Berkeley County because the citizens of southeastern Berkeley County felt they had to travel too far to the county seat of Martinsburg. Charles Washington, the founder of Charles Town and brother to George Washington, petitioned for a new county to be formed. It was named for Thomas Jefferson, author of the Declaration of Independence and third President of the United States. Virginia previously had a Jefferson County, which is now part of Kentucky. Accordingly, in the State records of Virginia, there are listings for Jefferson County from 1780 to 1792 and Jefferson County from 1801 to 1863, neither of which are still in Virginia.

The county's courthouse was the site of the trial for the abolitionist John Brown after his October 1859 raid on the federal armory in Harpers Ferry. Some 90 U.S. Marines serving under then Army Colonel Robert E. Lee and Lieutenants J.E.B. Stuart and Israel Greene put down the rebellion.

Brown was sentenced to death for murder, treason against the Commonwealth of Virginia, and conspiring with slaves to rebel. On December 2, 1859, John Brown was taken from the Charles Town jail a short distance to an open field and hanged. Among those attending the Brown execution was a contingent of 1500 cadets from Virginia Military Institute sent by the Governor of Virginia Henry A. Wise under the supervision of Major William Gilham and Major Thomas J. Jackson. In the ranks of a Richmond militia company stood John Wilkes Booth. Walt Whitman was also present.

The county was a frequent site of conflict during the Civil War, as Union and Confederate lines moved back and forth along the Shenandoah Valley. Some towns in the county changed hands between the Union and Confederacy over a dozen times, including Charles Town, and especially Harpers Ferry.

Jefferson County is the only part of modern-day West Virginia not exempted from the effects of the Emancipation Proclamation (as Berkeley County and the 48 counties designated as West Virginia had been). Slaves in the county thus were legally free as of January 1, 1863.

The Jefferson County Courthouse is the only courthouse in America to have held two treason trials: the trial of John Brown in 1859 and a trial arising from the Battle of Blair Mountain labor rebellion.

Jefferson County had voted for secession in the vote taken on May 23, 1861. However, Jefferson County, along with Berkeley County, both counties lying on the Potomac River in the Shenandoah Valley, with the consent of the Reorganized Government of Virginia voted in favor of annexation to West Virginia in 1863. Virginia tried to nullify this after the American Civil War, but the counties remained part of West Virginia.