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Chaetodipus nelsoni occurs on rocky substrates across the Mexican Altiplano. We investigated phylogeographic diversity within the species using morphologic, karyotypic, and molecular data. Data from nuclear (AFLP) and mitochondrial DNA support three distinct genetic groups with minimal substructuring coincident with biogeographic barriers previously identified in the Chihuahuan Desert and drainage basins of the Altiplano. We examined the morphological and karyotypic data in light of the molecular data. The results support recognition of three species within the currently accepted widespread C. nelsoni: 1) C. nelsoni restricted to a distribution centered on the El Salado River Basin; 2) elevation of C. n. collis to species, with two subspecies: one centered on Trans-Pecos Texas, the other on the Mapimí Basin (new subspecies); and 3) recognition of a new species, C. durangae, centered on the Nazas Basin and upper Río Mezquital drainage.