The pygmy rabbit (Brachylagus idahoensis), a leporid of the Great Basin and southern Wyoming, resides in habitats dominated by big sagebrush (Artemisia tridentata). Because of the patchy distribution of mature big sagebrush in southern Wyoming, we expected pygmy rabbits to exhibit genetic attributes associated with genetic isolation: high levels of spatially structured differentiation. We also predicted some reduction in genetic diversity in the peninsular Wyoming portion of the geographic range, relative to its Great Basin core. We used 14 microsatellite loci to compare genetic attributes between geographically distinct pygmy rabbit populations, and a subset of these microsatellite loci to compare with those of 2 sympatric cottontails (Sylvilagus spp.), both presumptive habitat generalists. Pygmy rabbits displayed moderate genetic diversity that was lower than that reported from locations near the core of the geographic range (Idaho and Montana). We observed only low levels of genetic differentiation in pygmy rabbits among sampling sites within Wyoming. Similarly, we observed low levels of differentiation in one species of cottontail sympatric with pygmy rabbits; however, the other species of sympatric cottontail displayed levels of differentiation congruent with those of populations at panmixia. Isolation-by-distance was the dominant genetic pattern observed, although examination of our data suggested that a 4-lane highway (Interstate 80) might affect gene flow measurably. In the recent evolutionary past, habitat connectivity and dispersal capacity for pygmy rabbits have been high enough to maintain gene flow among sites across southern Wyoming. Conservation of the species should focus on maintaining the connectivity among preferred habitats: old stands of big sagebrush.