Mitochondrial cytochrome-b (Cytb) and control region (CR) sequence data were used to investigate the phylogeography of the disjunct population of southern flying squirrels (Glaucomys volans) from Nova Scotia. Consistent with previous studies, there is little differentiation in Cytb sequences (0–1.3% sequence divergence) across the entire range of G. volans in North America. Nested clade phylogeographic analysis of CR sequences (which exhibited 0.5–5.7% sequence divergence) provided a finer scale of differentiation and allowed for inferences to be made regarding the phylogeographic history of the population from Nova Scotia. We propose that G. volans rapidly expanded its range from a southern refugium after the Wisconsin glaciation (∼12,000 years ago) and was then isolated in southwestern Nova Scotia during subsequent climactic fluctuations. More specifically, the population from Nova Scotia was isolated from extant populations in Ontario by the loss of intermediate populations. Although the population of G. volans in Nova Scotia would not be considered an evolutionarily significant unit under some definitions, examination of CR sequence data suggests that this population may nevertheless be diverging from other populations.