The montane sky islands of the Great Basin are characterized by unique, isolated habitats and communities that likely are vulnerable to extirpation with environmental change. A subspecies of yellow pine chipmunk, the Humboldt yellow pine chipmunk (Tamias amoenus celeris), is associated with the whitebark and limber pine forests of the Pine Forest Range (PFR) in Nevada. We sampled T. amoenus and least chipmunks (T. minimus) from the isolated PFR and compared genetic diversity between these populations and more “mainland” populations, including other subspecies of chipmunks. Given the high frequency of hybridization in Tamias, we tested for hybridization between T. amoenus and T. minimus in the PFR. We examined phylogenetic relationships, population divergence and diversity, and screened populations for a common pathogen, Borrelia hermsii, to gain insight into population health. We found T. amoenus of the PFR are closely related to T. amoenus in the Warner Mountains and Sierra Nevada, but maintain substantively lower genetic variation. Microsatellite analyses show PFR T. amoenus are highly genetically differentiated from other populations. In contrast, PFR T. minimus had higher genetic diversity that was comparable to the other T. minimus population we sampled. Pathogen screening revealed that T. amoenus carried higher pathogen loads than T. minimus in the PFR, although the prevalence of infection was similar to other Tamias populations. Our assessment of habitat associations suggests that the Humboldt yellow pine chipmunk almost entirely is restricted to the conifer systems of the PFR, while least chipmunks are prevalent in the other forests. Our work highlights the need for continued conservation and research efforts to identify how response to environmental change can be facilitated in isolated species and habitats.