Three cryptic species of the rodent Akodon (A. cursor, A. montensis and Akodon sp.) were analyzed. The two former species are sympatric in the Brazilian states of São Paulo, Rio de Janeiro and Minas Gerais, where hybrids have already been found. The third species, Akodon sp., occurs in an isolated area in Central Brazil. The identification of these species is difficult by the need of living animals. At present, karyotyping is the only method used in the identification of specimens. We used PCR-RFLP of the mitochondrial cytochrome gene to test the distinctiveness of the three species, which was confirmed by the absence of shared species-specific haplotypes. We also detected a geographical pattern of haplotypes distribution with highly polymorphic populations of A. cursor from Espírito Santo and of A. montensis from Rio Grande do Sul.