Phylogenetic relationships among 7 species of Reithrodontomys were examined using the complete mitochondrial cytochrome-b gene. DNA-sequence data were analyzed using parsimony (weighted and unweighted) and genetic distance methods. In all analyses, R. sumichrasti, R. megalotis, and R. zacatecae formed a monophyletic clade. Likewise, R. montanus and R. raviventris were depicted as sister taxa in all analyses. Our study supports recognition of R. zacatecae as a species distinct from R. megalotis and recognition of R. raviventris as distinct from R. montanus. Patterns of chromosomal evolution were examined using differential staining as resolved by 3 DNA probes and fluorescent in situ hybridization. Using the phylogeny based on DNA sequences, we concluded that there has been extensive chromosomal repatterning (in most cases karyotypic megaevolution) in the evolution of R. zacatecae, R. megalotis, R. sumichrasti, R. montanus, R. raviventris, and R. humulis.