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Landscape features such as rivers, mountains, desert basins, roads, and impermeable man-made structures may influence dispersal and gene flow among populations, thereby creating spatial structure across the landscape. In the US–Mexico borderland, urbanization and construction of the border fence have the potential to increase genetic subdivision and vulnerability to isolation in large mammal populations by bisecting movement corridors that have enabled dispersal between adjacent Sky Island mountain ranges. We examined genetic variation in black bears (Ursus americanus) from three regions in central and southern Arizona, US, to assess genetic and landscape connectivity in the US–Mexico border Sky Islands. We found that the three regions grouped into two subpopulations: the east-central subpopulation comprised of individuals sampled in the central highland and high desert regions, and the border subpopulation comprised of individuals sampled in the southern Sky Islands. Occupancy for the border subpopulation of black bears was influenced by cover type and distance to water, and occupancy-based corridor models identified 14 potential corridors connecting border Sky Island habitat cores with the east-central subpopulation. Biological quality of corridors, defined as length:width ratio and proportions of suitable habitat within corridors, declined with Sky Island dispersion. Our results show that black bears in the border subpopulation are moderately isolated from the east-central subpopulation, the main population segment of black bears in Arizona, and that connectivity for border bears may be vulnerable to anthropogenic activities, such as those associated with urbanization and trans-border security.