Cytogenetic studies were performed on 4 Anostomidae species from the Sapucai river, Minas Gerais, Brazil. Leporinus friderici, L. octofasciatus, L. striatus and Schizodon nasutus. Nuclear DNA content determination in 3 species revealed that L. striatus had a higher nuclear DNA content than S. nasutus and L. octofasciatus, respectively. Chromosome studies involving Giemsa, C-banding and Chromomycin A 3 staining showed that the chromosome number and morphology, of these 4 species was very similar. On the other hand, some differences regarding number of chromosomes with NORs in S. nasutus and some particularities of the pattern of heterochromatin distribution in L. friderici were observed. These data indicate the occurrence of some diversification of heterochromatin size and position and Ag-NORs reorganization of the karyotypes, as observed in some other species and populations of the family Anostomidae.