Peromyscine mice of the cricetid subfamily Neotominae comprise the most common and speciose assemblage of North American rodents. The composition and phylogenetic relationships within this group have been addressed using multiple character systems but remain unresolved, particularly the branching pattern of deeply rooted lineages. The relationships and status of several taxa formerly included within Peromyscus also are controversial. We present a new phylogenetic hypothesis based on combined mitochondrial and nuclear sequences using cytochrome b, interphotoreceptor retinoid-binding protein, and growth hormone receptor gene sequences, and include all major peromyscine lineages. Bayesian and parsimony approaches recover a congruent, well-resolved phylogeny, wherein Peromyscus forms a monophyletic assemblage only if a number of putative genera are included within it. Onychomys rather than Reithrodontomys is the sister taxon to Peromyscus (within which are included Habromys, Osgoodomys, Megadontomys, Podomys, and Neotomodon). Reithrodontomys in turn is the sister taxon to Isthmomys, and these 2 genera are the nearest outlier to Onychomys plus an expanded concept of Peromyscus, consistent with the recent recognition of this entire group as the tribe Reithrodontomyini. The Baiomyini, Ochrotomyini, and Neotoma (Neotomini) appear as successive outgroups to this clade. Based on these results, we present a systematic review of higher-level relationships among peromyscine rodents.