Phylogenetic relationships among 8 subspecies of Neotoma albigula and sister species from the United States and Mexico were examined using DNA sequence data from the mitochondrial DNA cytochrome-b gene. Parsimony, likelihood, and neighbor-joining analyses revealed a strong dichotomy between populations of N. albigula from Texas and eastern Mexico (eastern form) and those from New Mexico, Arizona, and northwestern Mexico (western form). These analyses indicate presence of 2 cryptic species within this taxon that are paraphyletic under current taxonomy. A sister-group relationship was found between N. albigula from Texas and eastern Mexico and N. micropus, whereas populations of N. albigula from New Mexico, Arizona, and northwestern Mexico formed a sister-group relationship with N. floridana. That latter group in turn formed a sister-taxon relationship to the Texas–eastern Mexico N. albigula and N. micropus clade. The Rio Grande and Rio Conchos seem to have been the major barriers restricting gene flow between ancestral populations of a N. floridana–like woodrat. Populations of N. floridana were further isolated geographically by reduction of suitable habitat brought about by changing climatic patterns that allowed formation of xeric plant communities soon after the end of the Late Wisconsin.