Wynkoop served as an officer in the First Colorado Volunteer Cavalry during the American Civil War, attaining the rank of major of volunteers, and was brevetted a lieutenant colonel in May 1865. During a period as post commander at Fort Lyon, Colorado in 1864, Wynkoop encouraged peace efforts with the Cheyennes, but was transferred in November 1864 to Fort Riley, Kansas, where he was posted at the time of the Sand Creek massacre. On behalf of the U.S. Army he investigated Col. John M. Chivington's conduct at Sand Creek, which led to Chivington's condemnation.