There is a crucial need to understand the genetic consequences of landscape modifications on continuous populations that could become fragmented, and to evaluate the degree of differentiation of isolated populations that were historically part of the core. Using 15 microsatellite loci, we evaluated the genetic structure of American black bears ( Ursus americanus Pallas, 1780) across a vast, contiguous Ontario landscape (>1 × 106 km2) that largely represents their pre-European settlement distribution. Because geographic barriers are absent, we predicted that isolation by distance would drive genetic structure. We identified three genetic clusters (Northwest, Southeast, and Bruce Peninsula) that were less differentiated than when assessed with mtDNA, suggesting the influence of male-biased dispersal on large-scale genetic differentiation. Isolation by distance (r = 0.552, P = 0.001) was supported by a weak, clinal variation between Northwest and Southeast, illustrating the challenges to delineate populations in wide-ranging taxa. The Bruce Peninsula cluster, confined to a small area under strong anthropogenic pressures, was more differentiated from neighbouring clusters (FST > 0.13, P < 0.0001), with a genetic diversity corresponding to disjunct populations of black bears. Our results could be used in landscape genetics models to project the evolution of population differentiation based on upcoming landscape modifications in northern regions of North America.