Intrageneric relationships within Noctilio were examined using the entire 1,140 base pairs of the mitochondrial cytochrome-b gene and 1,398 base pairs of the nuclear recombination activating gene-2. Sequences were analyzed to establish the relative time frame during which piscivory, the feeding strategy for hunting and eating fish, evolved in this genus. Piscivory represents the derived morphologic state for the genus Noctilio, and N. leporinus evolved from a N. albiventris-like ancestor. Distance values for specimens of N. leporinus from the Antilles, Central America, and South America suggest that N. leporinus speciated recently, 0.28–0.7 million years ago. Within N. albiventris, evidence exists that specimens from Peru diverged 1.16–2.9 million years ago. Analysis of these data suggests the possibility that 2 biological species are present within N. albiventris.