Levels of sequence variation in mitochondrial cytochrome-b gene were examined to ascertain if this molecule can provide a reference point in making decisions concerning species-level distinctions. DNA-sequence data from 4 genera of rodents (Neotoma, Reithrodontomys, Peromyscus, and Sigmodon) and 7 genera of bats (Artibeus, Carollia, Chiroderma, Dermanura, Glossophaga, Rhinophylla, and Uroderma), including recognized sister species, were examined to develop hypotheses for evaluating levels of sequence variation. Several patterns associated with DNA-sequence variation emerged from this study. Specifically, genetic distance values <2% were indicative of intraspecific variation; values between 2 and 11% had a high probability of being indicative of conspecific populations or valid species and merit additional study concerning specific status; and values >11% were indicative of specific recognition. It appears that genetic distance values may be useful for determination of species boundaries under the framework of the Genetic Species Concept.