The family Heteromyidae includes 6 genera of rodents traditionally placed in 3 subfamilies endemic to the Nearctic and northern Neotropical biogeographic regions. Although several of these taxa represent intensively studied members of North and Central American ecosystems (e.g., kangaroo rats and pocket mice), phylogenetic relationships within and among subfamilies, genera, and species-groups are not well understood. Here, we used maximum-likelihood, Bayesian, and maximum parsimony analyses of sequence data from 2 mitochondrial DNA genes, the cytochrome oxidase subunit 3 gene (699 base pairs [bp]) and the cytochrome-b gene (1,140 bp), to investigate phylogenetic relationships among 55 species-level taxa. We found robust support for monophyly of genera Dipodomys, Microdipodops, Chaetodipus, and Perognathus; sampling of Liomys and Heteromys was inadequate to evaluate their reciprocal status. All analyses converge on a phylogeny that robustly resolves several historically contentious issues, including monophyly of the subfamily Dipodomyinae (Microdipodops plus Dipodomys), and a monophyletic Chaetodipus that includes C. formosus, C. baileyi, C. rudinoris, and C. hispidus. However, Perognathinae (Perognathus plus Chaetodipus) is not supported, with no basal resolution among Perognathus, Chaetodipus, Dipodomyinae, and Heteromyinae. Many intrageneric clades receive strong support and are discussed herein. Although phylogenetic resolution is limited at the basal nodes of the Heteromyidae radiation, our results provide a basis for developing a provisional hypothesis regarding the historical biogeography in combination with independent information on the Neogene geological history of western North America and the fossil record of the family.