Dispersal and gene flow are significant components of metapopulation structure and dynamics. We examined gene flow in the pygmy rabbit (Brachylagus idahoensis), a sagebrush specialist that occurs in small, isolated populations in the Great Basin region and is believed to have limited dispersal abilities. We examined genetic diversity, gene flow, and population genetic structure, and investigated the effects of putative barriers to gene flow in 8 sample locations within 2 study areas in Idaho during 2002–2006. Potential barriers included secondary roads, highways, creeks, and agricultural pastures. Distance between sample locations ranged from 0.5 to 32 km. We expected that gene flow would be limited and that moderate levels of genetic structure would be apparent among sample locations separated by barriers and distances over several kilometers. However, analyses based on 15 microsatellite loci from 239 rabbits revealed low to moderate levels of genetic differentiation among sample locations. Bayesian cluster analyses suggested that sample locations separated by <14 km comprised 1 genetic cluster with high levels of gene flow among sample locations within clusters. We documented no significant evidence of a sex bias in dispersal based on sex-specific F_ST among sample locations, and relatedness (r), mean assignment index (mAI), and variance of assignment index (vAI) within sample locations; however, spatial autocorrelation suggested that juvenile females might disperse farther than males. These results indicate that dispersal capabilities of pygmy rabbits greatly exceed previous estimates, and that creeks and roads were not effective barriers to gene flow in our study areas.