Pleistocene climate fluctuations rearranged ecosystems, and influenced the contemporary distribution of modern species. Although specialist species were often restricted to isolated refugia by Pleistocene climate change, generalist species may have been less constrained in their distribution and movements. We used a combination of genetic data and previously published fossil data to investigate the phylogeography and contemporary population structure of a generalist species, the striped skunk (Mephitis mephitis). We sequenced a portion of the mitochondrial cytochrome-b gene (601 base pairs) and amplified 8 microsatellite loci from 314 striped skunk specimens. Phylogenetic analysis of the cytochrome-b gene revealed the presence of 4 distinct phylogroups, and examination of microsatellite data indicated a pattern of secondary contact among these clades. We infer from these data that during the Rancholabrean stage prior to the Illinoian glaciation, striped skunks emerged from a southern refugium in the Texas–Mexico region and colonized the southeastern United States, forming a 2nd, later refugium in the east. This colonization was followed by a 2nd dispersal event from the southern source population to west of the Rocky Mountains during the Illinoian glacial period. During the Sangamonian interglacial stage, 2 distinct subclades formed on either side of the Sierra Nevada. During the Holocene, the subclade that colonized the Great Basin then expanded east across the northern Rockies and recolonized the Great Plains to create an area of secondary contact with the southern phylogroup. Secondary contact occurred to a lesser extent with individuals from the eastern phylogroup east of the Mississippi River. It appears that periodic Pleistocene glacial expansions and retreats caused a series of range expansions and secondary contact events in this native North American species to create a complex pattern of population structure today.