He was made assistant quartermaster general on the staff of the Governor of Ohio on 17 April 1861, and during the Western Virginia Campaign acted as chief engineer of the Ohio troops. At the expiration of his three months' service he was appointed, on 15 August 1861, colonel of the 20th Ohio Infantry, and detailed as chief engineer of the Department of the Ohio, with charge of planning and constructing the defenses of Cincinnati. He was present at the Battle of Fort Donelson, where he led his regiment, and after the surrender was sent to north in charge of over 10,000 Confederate prisoners. At the Battle of Shiloh, he commanded the 3rd brigade of Maj. Gen. Lewis Wallace's division, but failing health compelled his retirement from active service, and he resigned on 19 April 1862. Whittlesey returned to service in September 1862, serving on Maj. Gen. Wallace's staff, providing assistance directing civilian and military labor for the defense of Cincinnati. Following the passing of the Confederate threat to the city, Whittlesey again returned to civilian life.