Pteronotus davyi, Davy's naked-backed bat, is a tropical bat whose current distribution in Mexico covers the watersheds of the Pacific and the Gulf coasts of Mexico, converges at the Isthmus of Tehuantepec, and continues into the Yucatan peninsula. We evaluated phylogeographic relationships among 18 populations in Mexico using data from the nucleotide sequence of the hypervariable II domain of the mitochondrial DNA control region from 105 specimens. Extant populations were distributed over 3 geographic regions (Pacific Coast, Gulf Coast, and Southeastern) that were delineated a priori according to floristic characteristics and biogeography. Coalescent simulations supported a phylogeographic model of 2 refugia situated in the south of Mexico (Arc and Soconusco refuges) during the late Pleistocene, followed by expansion into Pacific Coast, Gulf Coast, and Southeastern groups. The populations of the Southeast were genetically divergent from the Pacific and Gulf Coast populations, supporting the existence of 2 distinct lineages of P. davyi in Mexico, likely due to the Isthmus of Tehuantepec acting as a geographical barrier. Examination of our data revealed genetic differentiation of the Pacific and Gulf coastal groups but at a lower level relative to the Southeast. This pattern suggests that the Sierra Madre Occidental, Sierra Madre Oriental, and Sierra Madre del Sur act as physical barriers to dispersal for P. davyi.