Landscape-scale population genetic structure in vagile vertebrates was commonly considered to be a contradiction in terms whereas recent studies have demonstrated behaviour and habitat associated structure in several such species. We investigate whether landscape features influence morphological and genetic differentiation in a widespread, mobile raptor. To accurately describe genetic differentiation associated with regional landscape factors, we first investigated subspecies relationships at a continental scale. We used 17 microsatellite loci and five morphological measurements to investigate differentiation between eastern and western subspecies of red-tailed hawks (Buteo jamaicensis) and to identify patterns between differentiation and habitat within western North America. Bayesian and frequency-based analyses of microsatellite data revealed clear distinctions between B. j. borealis (eastern) and B. j. calurus (western) samples. Furthermore, hawks sampled in Texas were stouter than those collected from the Rocky Mountains and farther west. Among western samples, birds from the Great Basin, Rocky Mountains, and Washington were significantly different in morphology than those from Oregon and California. We identified a pattern of isolation by distance among western breeding sites around the Sierra Nevada. Given the long-range dispersal capabilities of raptors, this pattern suggests that population-specific habitat preferences, corresponding with habitat breaks between eastern and western slopes of the Sierra Nevada, and/or regionally variable population densities limit migration between the Mediterranean habitat of central California and the xeric habitats of southern California and interior west. We suggest habitat preferences and regionally disparate population densities may play a role in shaping genetic structure in vagile avian taxa.