Characidae is one of the largest fish families of the Neotropical region, and presenting a pronounced morphological variability, certainly does not constitute a monophyletic group. The cytogenetical data also show a large chromosomal variation and can provide important information for a better understanding of the relationships between the species of this group. 18S and 5S rDNA probes were used in the present study for the chromosomal mapping in different Characidae species from the São Francisco River (Astyanax lacustris, Astyanax scabripinnis, Hasemania nana, Piabina argentea, Orthospinus franciscensis, Serrapinnus heterodon, Serrapinnus piaba and Myleus micans) and Alto Paraná (Astyanax altiparanae) basins. Species with a single pair of chromosomes bearing the nucleolar organizing regions (NORs) were identified, as well as species with multiple NORs, up to a maximum of seven 18S rDNA sites. The number of 5S rDNA site was also not constant, varying from two to eight. The mapping of the ribosomal genes was useful for the characterization and differentiation of the analyzed species.