We evaluated evolutionary relationships of taxa within silky pocket mice of the Perognathus fasciatus species group, composed of 3 species, P. fasciatus, P. flavescens, and P. apache. These species are distributed throughout the Great Plains, Wyoming Basin, Colorado Plateau, and northern Chihuahuan Desert biogeographic regions in North America. We tested a previously postulated hypothesis of Pleistocene species divergence and introgression by analyzing mitochondrial DNA (mtDNA) and amplified fragment length polymorphisms (AFLPs). Both mtDNA and AFLP data support several genetic lineages in the fasciatus species group that are geographically structured. Molecular clock estimates reject a Pleistocene speciation hypothesis in favor of a deeper, more complex evolutionary history of initial divergence in the Miocene followed by secondary diversification beginning in the middle Pliocene and progressing through the Pleistocene. Results support recognition of an additional species within the group. Temporal and spatial congruence between the mtDNA clades in the fasciatus species group and other codistributed species of Perognathus appear to support a hypothesis of concerted diversification throughout the Chihuahuan Desert, Colorado Plateau, and Great Plains.