Northern yellow bats (Dasypterus intermedius) are tree-roosting bats in the family Vespertilionidae comprised of two subspecies: D. intermedius intermedius and D. intermedius floridanus. The two subspecies are thought to be geographically separated. Due to their cryptic morphology, this hypothesis has never been tested and can benefit from being examined within a molecular framework. In this study, mitochondrial sequence data from 38 D. intermedius and nuclear sequence data from 14 D. intermedius from across their range were used to test the hypothesis that genetically defined groups correspond geographically with the two morphologically defined subspecies. Although high levels of divergence of the mitochondrial sequence (11.6%) suggest genetically distinct clusters sympatric in southern Texas, no genetic structure was recovered with the nuclear marker. Moreover, the mitochondrial sequence data recovered a paraphyletic relationship between the two subspecies of D. intermedius with the Cuban yellow bat (D. insularis), whereas no such paraphyly was recovered from analysis of the nuclear marker. Divergence time based on analyses of mitochondrial sequence for the two subspecies was approximately 5.5 Ma. The patterns observed are hypothesized to be the result of past isolation of lineages and secondary contact that is currently contributing to gene flow.