Sagebrush-steppe ecosystems are among the most threatened terrestrial ecosystems in North America, but the plants and animals living on the Idaho National Laboratory (INL), a federally protected research facility located in the sagebrush steppe of southeastern Idaho, do not suffer from many of the deleterious effects of habitat conversion and fragmentation that characterize more typical western landscapes. This research describes basic patterns of gene flow among a group of relatively undisturbed Great Basin rattlesnake (Crotalus oreganus lutosus) hibernacula on the INL. Six polymorphic microsatellite DNA loci were used to genotype 200 individuals from 10 hibernacula located at spatial distances of 3–45 km from each other. Genetic connectivity, estimated using delta-mu squared ([δµ]²) and traditional F_ST calculations, is correlated with both Euclidean and least-cost path distances derived from a simple GIS-based denning habitat suitability model. Genetic analyses showed high levels of connectivity among rattlesnake hibernacula, but subtle differentiation between 2 distinct genetic clusters. Habitat suitability analyses produced significant correlations between gene-flow rates and the availability of intervening denning habitat. These findings suggest that the availability of high-quality thermoregulatory habitat along movement corridors helps maintain gene flow in a communally denning temperate snake species. Results from this study are interpreted within the context of an 18-year monitoring project on this population of snakes. These results also add a population genetic component to the existing body of natural history data pertaining to Great Basin rattlesnake population dynamics, reproduction, seasonal movements, hibernation, neonate survivorship, road effects, den modeling, and disturbance effects on the INL.