The phylogeography of the American black bear (Ursus americanus) is characterized by isolation into glacial refugia, followed by population expansion and genetic admixture. Anthropogenic activities, including overharvest, habitat loss, and transportation infrastructure, have also influenced their landscape genetic structure. We describe the genetic structure of the American black bear in the American Southwest and northern Mexico and investigate how prehistoric and contemporary forces shaped genetic structure and influenced gene flow. Using a suite of microsatellites and a sample of 550 bears, we identified 14 subpopulations organized hierarchically following the distribution of ecoregions and mountain ranges containing black bear habitat. The pattern of subdivision we observed is more likely a product of postglacial habitat fragmentation during the Pleistocene and Holocene, rather than a consequence of contemporary anthropogenic barriers to movement during the Anthropocene. We used linear mixed-effects models to quantify the relationship between landscape resistance and genetic distance among individuals, which indicated that both isolation by resistance and geographic distance govern gene flow. Gene flow was highest among subpopulations occupying large tracts of contiguous habitat, was reduced among subpopulations in the Madrean Sky Island Archipelago, where montane habitat exists within a lowland matrix of arid lands, and was essentially nonexistent between two isolated subpopulations. We found significant asymmetric gene flow supporting the hypothesis that bears expanded northward from a Pleistocene refugium located in the American Southwest and northern Mexico and that major highways were not yet affecting gene flow. The potential vulnerability of the species to climate change, transportation infrastructure, and the US–Mexico border wall highlights conservation challenges and opportunities for binational collaboration.