Fragmentation of wildlife populations can have detrimental effects, including genetic differentiation of populations, loss of genetic diversity, and inbreeding depression. We evaluated the genetic structure among isolated colonies of black-tailed prairie dogs (Cynomys ludovicianus) along an urban gradient in southern Denver, Colorado. Urban colonies are important ecologically and for educational purposes, and they serve as source populations for relocation efforts. Levels of genetic differentiation between colonies were high relative to colonies in natural habitat at comparable or greater distances. Prairie dog colonies depend on dispersal among colonies for long-term persistence, and we found evidence for reduced but measurable rates of movement of individual prairie dogs among urban fragments. We observed a trend for smaller and more isolated colonies to exhibit lower genetic diversity, but we did not detect inbreeding in any of the colonies sampled. Isolation-by-distance measures, including measures based on permeability of various features of urban habitat such as roads and development, did not explain genetic differences. Our system represents a possible end point in the genetic consequences of continued loss and isolation of prairie dog colonies as fragmentation increases in both urban and natural landscapes. Urban development could affect dispersal in unexpected and complex ways and requires further study, but prairie dog colonies and their associated wildlife communities in urban areas have the potential for long-term persistence if not extirpated by human activity.