At the beginning of the American Civil War in 1861, Wistar chose to follow his home state and the Union cause. He raised a company of men and was elected its captain. Wistar's company was added to the 71st Pennsylvania Infantry, originally known as the California Regiment. This regiment was organized at Fort Schuyler in New York. On June 28, Wistar was promoted to the rank of lieutenant colonel, and on July 1 the 71st left for Fortress Monroe on the Virginia Peninsula. On July 22, Wistar and the 71st was then ordered to Washington, D.C., forming part of the capital's defenses until that fall. Wistar participated in the much-publicized Union defeat in the Battle of Ball's Bluff on October 21. In the fight, he temporarily led the regiment and was seriously wounded, hit in his right elbow, his jaw, and thigh.