This regiment organized at Camp Goldthwaite near Talladega, Alabama, on April 16, 1862. Within a few weeks the regiment was ordered to report and Chattanooga TN, and subsequently joined Confederate forces in operations against Federal forces near Cumberland Gap. After the enemy evacuated that point, the 31st Alabama marched into Kentucky with Stevenson's Division, but returned without making contact with Union troops.

During the late fall of 1862, Stevenson's Division was ordered to Mississippi. The regiment was placed in an Alabama brigade commanded by Brig. Gen. Edward D. Tracy, who was killed when his brigade fought the advance of Grant's army at Port Gibson, MS on May 1, 1863. The regiment lost its flag during the decisive battle at Champion Hill, May 16, 1863. It is one of the few Alabama flags still retained by a northern state (Iowa). The regiment lost a second brigade commander when Isham Garrott was killed in the trenches of the Square Fort at Vicksburg MS on June 17, 1863. Survivors of the siege surrendered on July 4, 1863, and were released to go to exchange camps a few days later.

Now led by Brig. Gen. Edmund W. Pettus, the brigade was issued new arms and equipment and sent to the Army of Tennessee outside Chattanooga TN. Some of the brigade was involved in the fighting on Lookout Mountain, Nov. 24, 1863, all the brigade participating in the defense of Missionary Ridge the following day. Officers and men withdrew with the army to winter camp at Dalton GA.

Pettus' Brigade continued to serve in Stevenson's Division during the Atlanta Campaign. The brigade saw action at Rocky Face, Resaca, New Hope Church, Big Shanty and Kolb's Mill. It was also involved in the Battle of Atlanta, July 22, 1864, and the Battle of Jonesboro GA, Aug. 31-Sept. 1, 1864. During the Tennessee Campaign, Pettus led his command during the crossing of the Duck River at Columbia TN, the disastrous battle of Nashville TN, Dec. 15-16, 1864, and served with the rear guard as the army withdrew from Tennessee.

April 9, 1865, survivors of the regiment became companies “G”, “H”, and “K” of the consolidated 23rd Alabama Infantry Regiment. This command surrendered at Salisbury, North Carolina, on May 2, 1865.