Populations of flying squirrels from the Mesoamerican highlands represent the least understood members of the genus Glaucomys. Traditionally, these populations have been considered to be southern disjuncts of the southern flying squirrel (G. volans), a species that is widespread across the deciduous and mixed-deciduous forests of eastern North America. The limited number of museum specimens of Mesoamerican flying squirrels has made discerning the systematic and biogeographic relationships of these populations a challenge. We used ancient DNA techniques to extract, amplify, and sequence a 571-base pair segment of the mitochondrial DNA cytochrome-b gene from 22 of 34 available museum specimens. Mesoamerican flying squirrel data were combined with homologous sequences from representative populations of Glaucomys from the United States and Canada. This combined data set was analyzed using maximum-likelihood and Bayesian methods. Results indicate that G. volans is monophyletic and contains 2 monophyletic subclades, 1 from Mesoamerica and the other from eastern North America. Our results have important implications regarding the nature of the historical biogeographic connection between the temperate biotas of Mesoamerica and eastern North America. The divergence of populations of G. volans in eastern North America from those in Mesoamerica appears to have occurred in the middle Pleistocene (approximately 0.75–0.5 × 10⁶ years ago), considerably earlier than a late-Pleistocene connection previously hypothesized. Our analyses also show that populations of G. volans from eastern North America exhibit a clear signature of recent, rapid population expansion and that Mesoamerican populations of G. volans exhibit higher levels of genetic variability than those found across eastern North America. The documentation of substantial genetic diversity and population structure in Mesoamerican populations of G. volans is especially noteworthy because these populations face ongoing habitat loss due to human activities. Anthropogenic habitat degradation of the high-elevation forests these mammals inhabit likely will be exacerbated by global climate change. Therefore, we suggest that the conservation status of Mesoamerican flying squirrels be considered data deficient at a minimum with a high potential for future studies to reveal that many populations are near threatened or vulnerable.