Karyotypic analyses were performed in fishes from the genus Hoplias (H.malabaricus and H. lacerdae groups) from the Sao Francisco River basin (Brazil), in an impacted region by a river transposition which altered the local ecology and fish fauna. The karyotypes were investigated using chromosomal markers obtained from classic and molecular cytogenetics (Giemsa, CMA3 and DAPI staining, C-banding, Ag-NORs, and FISH with 18S rDNA, 5S rDNA and 5SHindIII satellite DNA probes). Two karyotypic forms were found for the H. malabaricus group—karyomorph F, corresponding to the native form from the Sao Francisco River basin, and karyomorph A, corresponding to the invading form from the Upper Parana River basin. Specimens from the H. lacerdae group exhibited striking chromosome differences in relation to the H. malabaricus group, thereby enabling good cytotaxonomic characterization and inferences regarding the karyotype evolution of these groups.