The potential for widespread, mobile species to exhibit genetic structure without clear geographic barriers is a topic of growing interest. Yet the patterns and mechanisms of structure—particularly over broad spatial scales—remain largely unexplored for these species. Bobcats occur across North America and possess many characteristics expected to promote gene flow. To test whether historical, topographic or ecological factors have influenced genetic differentiation in this species, we analysed 1 kb mtDNA sequence and 15 microsatellite loci from over 1700 samples collected across its range. The primary signature in both marker types involved a longitudinal cline with a sharp transition, or suture zone, occurring along the Great Plains. Thus, the data distinguished bobcats in the eastern USA from those in the western half, with no obvious physical barrier to gene flow. Demographic analyses supported a scenario of expansion from separate Pleistocene refugia, with the Great Plains representing a zone of secondary contact. Substructure within the two main lineages likely reflected founder effects, ecological factors, anthropogenic/topographic effects or a combination of these forces. Two prominent topographic features, the Mississippi River and Rocky Mountains, were not supported as significant genetic barriers. Ecological regions and environmental correlates explained a small but significant proportion of genetic variation. Overall, results implicate historical processes as the primary cause of broad-scale genetic differentiation, but contemporary forces seem to also play a role in promoting and maintaining structure. Despite the bobcat’s mobility and broad niche, large-scale landscape changes have contributed to significant and complex patterns of genetic structure.