Phylogenetic relationships of 6 species of the genus Akodon have been determined by mtDNA sequence analysis of the cytochrome-b gene, with the majority of specimens also identified by karyotype. We used maximum parsimony and distance analyses of 54 sequences to assess phylogenetic relationships and minimum spanning trees to compare genetic and geographic relationships among populations. The genus Bolomys was used as the outgroup. Akodon cursor, A. aff. cursor, and A. montensis, 3 morphologically cryptic but karyotypically different species of the cursor complex, each formed reciprocally monophyletic lineages. Twenty-one haplotypes were present in 23 individuals of A. cursor, and each of the 16 specimens of A. montensis had a unique haplotype. Within species, populations of A. cursor (19 localities) showed high variation with little geographic pattern. Distant populations (∼750 km apart, differing by 12 nucleotides) exhibited similar levels of differentiation as did geographically close populations (∼150 km apart, differing by 17 nucleotides). In contrast, A. montensis (12 localities) was structured geographically and exhibited a pattern consistent with an isolation-by-distance model of differentiation. Among other species in southeastern Brazil, A. mystax was related most closely to the cursor species complex, A. lindberghi was somewhat more distant, and A. serrensis was quite distinct.