Phylogenetic relationships among 4 subspecies of Neotoma floridana 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 support the recognition of N. f. magister as a distinct species (N. magister). Populations of eastern woodrats from the southern and western United States also possessed relatively high levels of sequence divergence (5.3%), which may be indicative of ongoing divergence between these populations. These findings support conclusions from previous studies and add clarity to the biogeographic processes influencing evolution and diversification of this group of rodents.