Chromosomal characteristics of Hoplerythrinus unitaeniatus populations from 5 Brazilian river basins, namely Arinos (Amazonas basin), Araguaia, Paraguai, Alto Paraná, and São Francisco were analyzed by conventional Giemsa staining, C-banding, silver nitrate impregnation, and fluorescence in situ hybridization (FISH) with 18S and 5S rDNA and telomeric sequence (TTAGGG)n probes. The diploid chromosome number was 2n = 48 in representatives of the populations from Paraguai and Alto Paraná River basins and 2n = 52 for those from the Arinos and Araguaia River basins. The São Francisco population had individuals with 2n = 50 and 52 occurring in sympatry. C-banding showed heterochromatic blocks mainly located at interstitial and pericentromeric positions in most of the chromosomes. Silver nitrate impregnation demonstrated simple NORs for representatives from Arinos and Araguaia River populations and multiple NORs for specimens from Paraguai, Alto Paraná, and São Francisco River populations. FISH with 18S and 5S rDNA probes revealed many chromosomes carrying these cistrons, with up to 21 chromosomes bearing 18S rDNA sites (Alto Rio Paraná basin) and up to 12 chromosomes with 5S rDNA sites (Paraguai basin), besides the occurrence of colocalization in all populations. FISH with telomeric sequence (TTAGGG)n detected sites in the terminal portion of the chromosomes in all populations. These data reinforce the idea that H. unitaeniatus is a species complex. Evolutionary and biogeographical aspects of the group in the Neotropical region are discussed.