Phylogenetic relationships among 15 individuals representing 6 subspecies of Neotoma mexicana from the United States and Mexico were examined using DNA sequences from the mitochondrial DNA cytochrome b gene. Parsimony, likelihood, and genetic distance analyses revealed a dichotomy between populations of N. mexicana from the United States and northern Mexico and those from south of the Trans-Volcanic belt in southern Mexico. A 2nd dichotomy existed between wood rats in the Sierra Madre del Sur in southwestern Mexico and those south of the Isthmus of Tehuantepec. Further, populations of N. mexicana from the United States and those from Mexico possessed relatively high levels of sequence divergence indicating substantial genetic differentiation between these 2 groups. These data indicate that the 3 taxa probably represent sibling species and that consideration should be given to the elevation of the southern and southwestern Mexico forms to specific status. Additionally, paleontological material from Pleistocene wood rat middens in New Mexico and Mexico provided evidence for the effects of climatic changes on this group of wood rats and the means for assessing the phylobiogeography of the N. mexicana species group.