Understanding the causes and consequences of reduced gene flow remains a primary goal of landscape genetics. In this study we tested two hypotheses that potentially explain genetic differentiation across the southern portion of the range of the pygmy rabbit. The first hypothesis is that extensive pluvial lakes that existed during the Pleistocene are primarily responsible for modern patterns of genetic subdivision. The second hypothesis is that the modern distribution of sagebrush is the primary determinant of genetic differentiation across this portion of the pygmy rabbit’s range. We calculated least-cost paths, cost-weighted distance, and cumulative current between thirteen populations of pygmy rabbit. Our results show strong support for the sagebrush hypothesis with over 70% of genetic differentiation explained by the modern distribution of sagebrush, while the pluvial lake hypothesis explained relatively little variation. Especially in central and eastern Nevada, our analyses suggest that multiple pathways of connectivity through sagebrush may enhance gene flow. In contrast, the Mono Basin populations of pygmy rabbit are genetically distinct and largely isolated because of lack of sagebrush habitat between this region and the more contiguous range. Our results highlight the importance of continuous sagebrush habitat in maintaining genetic connectivity in pygmy rabbit.