We present the most comprehensive systematic study to date of Necromys, a rodent genus distributed in open areas north and south of Amazonia and in Andean grasslands. The study is based on sequences of the cytochrome-b gene that were analyzed by parsimony and Bayesian approaches. The analyses include sequences of 62 specimens from 51 localities from Argentina, Bolivia, Brazil, Colombia, Paraguay, Peru, Uruguay, and Venezuela, representing all but 1 of the species currently recognized in the genus. Necromys was recovered as a monophyletic group and we found a large polytomy at its base that involves 3 lineages. One, represented by the Andean N. lactens, shows a marked phylogeographic pattern. The 2nd clade is formed by N. urichi from the northern grasslands of South America and N. amoenus from the central Andes. Results suggest that each of these taxa may represent more than 1 biological species. The 3rd clade is formed by lowland species found south of Amazonia. Within this clade N. obscurus is sister to the remaining species. Haplotypes recovered from specimens assigned to N. benefactus, N. temchuki, and N. lasiurus form a clade, but these taxa do not form reciprocally monophyletic groups, nor does this large clade possess geographic structure. These genealogical results, discussed in the context of genetic variation, are the basis of taxonomic (e.g., N. benefactus and N. temchuki are regarded as junior synonyms of N. lasiurus) and biogeographic considerations.