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Historically, silky desert mice of the genus Eligmodontia have presented mammalogists with a complex and somewhat vexing taxonomic problem. At various times silky desert mice have been assigned to several species or to only one or two species. Most recently karyological evidence has suggested the presence of at least two morphologically cryptic species, E. morgani and E. lypus in Patagonia. To further elucidate this issue, we used a combination of karyological data and DNA sequences from the mitochondrial cytochrome b protein-coding gene to test the hypothesis that there are at least two genetically distinguishable and reproductively isolated species in Patagonia. By this means we showed that E. morgani and E. typus can be recognized readily by their karyotypes and mtDNA. In fact they exhibit more than 10% divergence in a 348-base pair region of cytochrome b. Within each species the mtDNA sequences enabled us to identify numerous maternal lineages. Results of PAUP analyses used to place these lineages in a geographic context suggested that the oldest lineage in the sample of each species was rare and occurred in the northwestern portion of the study area. Each species also was characterized by a common, geographically widespread ’’star’’ lineage. Typically at each locality we found the star lineage and one or more local, related lineages. These data are consistent with a historical bottleneck, rapid expansion through the star lineage, and subsequent settling in and production of new lineages. The unequivocal identification of individuals based on mtDNA, not previously possible on the basis of morphology alone, allowed us to begin mapping distribution of these two largely allopatric species. The data suggest that E morgani occurs in more mesic habitats, whereas E. typus occupies more arid areas mostly to the east of the range of E. morgani.