The genus Nyctinomops comprises 4 species of bats in the free-tailed family Molossidae: Nyctinomops macrotis, Nyctinomops femorosaccus, Nyctinomops aurispinosus, and Nyctinomops laticaudatus. The objective of this study was to use mitochondrial sequence data to test hypotheses of relationship within Nyctinomops. A total of 460 bases of cytochrome b and 686 bases of NADH dehydrogenase were sequenced and analyzed from 22 individuals by use of parsimony, maximum likelihood, and Bayesian methods. Sequences from other molossid genera (Tadarida, Cynomops, and Otomops) were used as outgroups. In addition, a series of Nyctinomops specimens from Peru was re-assessed using morphological and molecular data. Our results agree with previous studies using morphological similarity in the placement of the largest member of the genus, N. macrotis, as sister to the remaining species. Furthermore, both mitochondrial data sets recovered the following relationship for the other members of the genus: (N. aurispinosus (N. laticaudatus, N. femorosaccus)).