Three species of Mexican deer mice of the Peromyscus mexicanus species group (P. grandis, P. guatemalensis, and P. zarhynchus) were characterized morphologically and genetically to test hypothesized concepts of species limits. We investigated if previously proposed phenetic relationships among these 3 taxa were supported by morphometric and genetic data. Analyses of nongeographic and geographic variation for individuals from 36 localities in Guatemala and southeastern Mexico were conducted to assay morphologic and geographic boundaries. In addition, 35 mitochondrial cytochrome-b gene sequences were analyzed using maximum-parsimony and Bayesian inference methods to determine relationships among the 3 taxa. This study based on comparisons to type specimens provided support for the presence of 3 morphologically and genetically distinct units. Our analyses suggest that P. grandis and P. guatemalensis are more closely related to each other than either is to P. zarhynchus, rejecting existing hypotheses that suggest that P. zarhynchus and P. grandis are phenetically more similar. The results of this study depict relationships among other members of the P. mexicanus group and patterns of speciation and biogeography and allow identification of regionally important phylogeographic units in Mesoamerica.