Many leporid species have overlapping ranges, but ecological differences may make for profoundly different population structure in the same area. In southern Illinois, swamp rabbits (Sylvilagus aquaticus) and eastern cottontails (S. floridanus) co-exist, but swamp rabbits are habitat specialists associated with floodplain forests and eastern cottontails exhibit more general habitat preferences. Additionally, swamp rabbits exist at the northern edge of their range in southern Illinois, whereas eastern cottontails are well within the core of their range. To determine whether these differences resulted in differences in genetic structure, we assessed the population differentiation of these 2 sympatric species in southern Illinois using polymorphic microsatellite markers. We employed a combination of sampling techniques including tissue sampling from trapped rabbits and collection of fecal pellets from swamp rabbit latrine logs. Swamp rabbits appeared to be differentiated within 50 km of Illinois’ Cache River watershed (K = 4 populations) suggesting that local populations are relatively isolated, whereas eastern cottontails exhibited no discernable genetic structure (K = 1). Such a result confirms the expected relationship of greater genetic structure in a habitat specialist (i.e., swamp rabbits) versus a habitat generalist (i.e., eastern cottontails). Unlike eastern cottontails, our results suggest that swamp rabbits experience low genetic connectivity in southern Illinois likely due to restricted dispersal, linear distribution of habitat, and smaller effective population size as mediated by habitat fragmentation.