Knowledge of the distribution of genetic resources within and among taxa is prerequisite for development of management strategies which facilitate conservation of those resources. We used restriction-site associated DNA (RAD) sequencing to survey genetic variation in Alvord Chub and Borax Lake Chub from the Alvord Basin in southeastern Oregon and northern Nevada, USA. Our specific goals were to gain an understanding of the population genetic structure of Alvord Chub and the relationship between this species and Borax Lake Chub. Despite the fact that our collections were taken from ponds and streams, isolated from one another by kilometers of desert, our results revealed that diversity in Alvord Chub was primarily distributed among two regional groupings, which we hypothesize to be related to habitat changes following the end of the most recent glaciation event, approximately 10 thousand years ago. Our results further revealed that Alvord Chub and Borax Lake Chub shared common ancestors during that glaciation event, and that divergence between the two may have been sympatric. Finally, we observed evidence that the clade containing Alvord Chub and Borax Lake Chub was isolated from its closest extant relative, the Tui chub, during the Miocene Epoch, when volcanic flows were forming the mountains surrounding what became the Alvord Basin. These results will be useful for informing conservation strategies for Alvord Chub, and provide new insights regarding the role of these species in the evolutionary legacy of the region.