Sixteen populations of Peromyscus on islands in the Sea of Cortéz (= Gulf of California), Mexico, were compared with 9 mainland species of Peromyscus based on sequence data for a 699-base-pair fragment of the mitochondrial DNA (mtDNA) COIII gene. An unrooted neighbor-joining tree based on corrected pairwise estimates of sequence divergence among variable mtDNA haplotypes indicated a recent (late Pleistocene) origin from a source on the adjacent mainland for 10 island forms representing P. boylii, P. crinitus, P. eremicus, P. eva, P. fraterculus, and P. maniculatus. Five other populations did not seem to be derived from species currently on the nearest mainland, suggesting overwater dispersal or distributional changes on the mainland after drowning of land-bridge connections. One population, possibly of more ancient origin, on Isla Cerralvo near the Baja California peninsula, probably originated via trans-Gulf rafting from the Sonoran mainland. Based on these results, 4 insular species (P. stephani, P. interparietalis, P. caniceps, and P. dickeyi) should be considered subspecies of P. boylii, P. eremicus, P. fraterculus, and P. merriami, respectively. The emergent view of evolutionary relationships within the subgenus Haplomylomys in the region reflects pre-Pleistocene phylogeographic events on the mainland surrounding the Gulf and a more recent origin of island populations.