We examined patterns of genetic variation and diversity of extant pygmy rabbit (Brachylagus idahoensis) populations across the species' current range in Nevada and California. Our aims were to determine population genetic structure and levels of diversity across the southern portion of the species' range. We genotyped 13 microsatellite loci from 194 fecal samples collected across 14 localities. Our Bayesian cluster analyses found 2 genetically distinct groups: 1 in the Mono Basin of California and the other encompassing all remaining Nevada Great Basin populations. Considering only the Nevada Great Basin group, we found 4 minimally divergent groups that overlap spatially with many individuals maintaining composite genomes with representation from multiple genetic groups. On average, the Mono Basin populations maintain lower levels of genetic diversity than the Nevada Great Basin populations as measured by both heterozygosity and numbers of alleles. The Mono Basin contains the only populations of pygmy rabbits remaining in California. Given their geographic isolation from other known populations and their genetic distinction, these remnant, peripheral populations warrant special attention to ensure maintenance of genetic variation and continued viability.