The desert pocket mouse (Chaetodipus penicillatus) comprises 6 nominate subspecies that occupy warm, sandy desert-scrub habitats across the Mojave and Sonoran deserts. The most thorough morphological assessment within the species noted variable levels of distinctiveness, leading to uncertainty regarding the geographic distributions of subspecies. Subsequent genetic assessments using chromosomal, allozymic, and mitochondrial DNA (mtDNA) sequence data detected a general east–west divergence centered on the Colorado River, but few locations were included in these assessments. We investigated phylogeographic structure in C. penicillatus by sequencing regions of mtDNA for 220 individuals from 51 locations representing all continental subspecies. We identify 2 major monophyletic mtDNA lineages (clades) roughly centered in the Mojave and Sonoran deserts. These clades broadly overlap along the Lower Colorado River valley and adjacent desert regions across most of the range of C. p. penicillatus. Outside this zone of mtDNA clade overlap, Sonoran clade haplotypes occur in populations from across the range of C. p. pricei and extend to the northwestern edge of the Sonoran Desert within the southern range of C. p. angustirostris. Northern clade haplotypes occur in populations within the ranges of C. p. sobrinus and C. p. stephensi and in populations from the western Mojave Desert in the northern range of C. p. angustirostris. Based on rough estimates for rates of sequence evolution, divergence among the major clades appears to have occurred during the Pleistocene, but well before the latest glacial maximum. The secondary contact among the major clades appears to have some longevity, with little evidence of recent, postglacial range expansion. We develop ecological niche models (EMNs) for the major lineages of C. penicillatus, and project these models onto reconstructions of climatic conditions during the latest glacial maximum (LGM; 18,000–21,000 years ago). The ENMs for each clade indicate differences in predicted current geographic distributions as well as distributions during the LGM. Models for the LGM indicate broad retention of potential habitat within the area of contact among the major clades. Furthermore, the ENM for the Mojave clade in particular indicates retention of suitable habitat during the LGM in small isolated patches within northern areas, consistent with the haplotype network that supports the perspective that some populations from the Mojave clade were isolated within northern refugia during the last glacial period.