After the state of Mississippi seceded just before the start of the Civil War, Barksdale resigned from Congress to become adjutant general, and then quartermaster general, of the Mississippi Militia, at the rank of brigadier general, with date of rank March 1, 1861. On May 1, he was appointed colonel in the Confederate States Army of the 13th Mississippi Infantry, a regiment that he led in the First Battle of Bull Run that summer, and the Battle of Ball's Bluff in October. The following spring, he took his regiment to the Virginia Peninsula and fought in the Peninsula Campaign and the Seven Days Battles. When his brigade commander, Brig. Gen. Richard Griffith, was mortally wounded at the Battle of Savage's Station on June 29, 1862, Barksdale assumed command of the brigade and led it in an heroic, but bloody and futile, charge at the Battle of Malvern Hill. The brigade became known as "Barksdale's Mississippi Brigade." He was promoted to brigadier general on August 12, 1862.