We assessed the genetic status of two endangered subspecies of the northern flying squirrel (Glaucomys sabrinus) that are restricted to isolated stands of high elevation spruce-fir and adjacent spruce-fir-hardwood ecotonal habitat in the Appalachian Mountains of eastern North America. We used mitochondrial DNA (mtDNA) and allozyme data to estimate levels of genetic variability in the two subspecies of interest and then evaluated this information in the context of large-scale phylogeographical structure and overall genetic variability for the entire species and for the closely related and partially sympatric southern flying squirrel (Glaucomys volans). This broader analysis involves much of North America's northern coniferous forest biome, together with the deciduous forest biome of eastern North America. Our results support the evolutionary distinctness of the endangered Appalachian populations of G. sabrinus. These populations possess several private alleles and have levels of genetic variability that are substantially lower than those observed in conspecific populations found elsewhere. However, the endangered Appalachian populations of G. sabrinus have higher levels of genetic variability than those observed in populations of G. volans from across eastern North America. These results highlight the utility of evaluating the conservation genetics of small and isolated populations within a broad-scale comparative evolutionary and biogeographical framework.