The cytochrome-b gene was sequenced and data were analyzed for 104 Perognathus from localities in Mexico, Arizona, California, New Mexico, Oklahoma, and Texas to evaluate the validity of P. flavus and P. merriami and to assess the occurrence of species of Perognathus in Oklahoma. Unweighted-parsimony, minimum-evolution, maximum-likelihood (TrN I G), and Bayesian analyses revealed seven well-supported terminal clades corresponding to currently recognized species of Perognathus. Results validate recognition of P. flavus and P. merriami as distinct species, document the occurrence of P. merriami within Oklahoma, and reveal a second potential contact zone between P. flavus and P. merriami in the Oklahoma Panhandle.