The family Cichlidae is one of the most species-rich taxa in the Neotropics. However, the factors that determine these high levels of biodiversity remain unexplored. We have analyzed the morphological, cytogenetic, and molecular data from 62 specimens of a widespread cichlid, Geophagus brasiliensis, from three adjacent basins in southeastern Brazil. Morphological analyses did not show differences among specimens. The cytogenetic data indicate the occurrence of multiple nucleolar organizer regions and four sympatric karyotypes that differ in the first pair of chromosome morphology, in the Doce River Basin; whereas the karyotype from the Paraíba do Sul Basin is widely divergent. The molecular data--616 bp fragment of cytochrome oxidase subunit I--revealed two haplogroups with the deepest genetic divergence (6.4%) ever reported within a nominal species in the Neotropical Region: One of the haplogroups is restricted to the quaternary lakes in the middle portion of the Doce Basin and the Mucuri River, whereas the other haplogroup is composed of haplotypes from elsewhere in the Doce Basin and the Paraíba do Sul Basin. These patterns suggest that G. brasiliensis undergoes a cryptic speciation process involving three major lineages that differ from the African explosive cichlid radiation.