In this study, 43 specimens of Hoplerythrinus unitaeniatus from the São Francisco River basin were chromosomally analyzed by conventional Giemsa staining, C-banding, silver nitrate impregnation, and fluorescence in situ hybridization (FISH) with probes of 5S and 18S rDNA. The diploid numbers found were 50 and 52 chromosomes, showing the existence of two well-defined biological entities in sympatry. Specimens with 51 chromosomes, which showed three distinct karyotypic forms, were also found and are characterized as natural hybrids due to the correspondence with the chromosomes of the specimens with 50 and 52 chromosomes. By FISH using 5S and 18S rDNA probes, it was possible to detect specific chromosomal markers for the specimens with 50 and 52 chromosomes, as well as the occurrence of common sites in both. The specimens with 51 chromosomes showed intermediate patterns for these markers, reinforcing the hypothesis that these are actual natural hybrids. A review and new classification for the karyomorphs of H. unitaeniatus have also been proposed.