Potential declines in native pollinator communities and increased reliance on pollinator-dependent crops have raised concerns about native pollinator conservation and dispersal across human-altered landscapes. Bumble bees are one of the most effective native pollinators and are often the first to be extirpated in human-altered habitats, yet little is known about how bumble bees move across fine spatial scales and what landscapes promote or limit their gene flow. In this study, we examine regional genetic differentiation and fine-scale relatedness patterns of the yellow-faced bumble bee, Bombus vosnesenskii, to investigate how current and historic habitat composition impact gene flow. We conducted our study across a landscape mosaic of natural, agricultural and urban/suburban habitats, and we show that B. vosnesenskii exhibits low but significant levels of differentiation across the study system (FST = 0.019, Dest = 0.049). Most importantly, we reveal significant relationships between pairwise FST and resistance models created from contemporary land use maps. Specifically, B. vosnesenskii gene flow is most limited by commercial, industrial and transportation-related impervious cover. Finally, our fine-scale analysis reveals significant but declining relatedness between individuals at the 1–9 km spatial scale, most likely due to local queen dispersal. Overall, our results indicate that B. vosnesenskii exhibits considerable local dispersal and that regional gene flow is significantly limited by impervious cover associated with urbanization.