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AbstractAmong the more than 30 families of the order Siluriformes, Heptapteridae is composed of 189 species distributed into 24 genera. Rhamdia, which has wide distribution throughout the Neotropical region, presents only 11 valid species, with 8 being in the Brazilian territory. Rhamdia quelenis the only species considered as widely distributed in almost all Brazilian hydrographic basins. It is a Neotropical fish species with a confusing taxonomical history. Classic and molecular cytogenetics data for three populations of this species from two large Brazilian hydrographic basins (Paraná and Araguaia) are presented here. The diploid number found for the three analyzed populations was 58 chromosomes, but with distinct karyotypic formulae. The presence of B chromosomes was detected in the two Araguaia River populations with intra- and interindividual variation. C-banding evidenced little heterochromatin in the three analyzed populations. FISH with 18S rDNA probes evidenced a single chromosome pair bearing this site, confirming the presence of simple NORs, as visualized through silver nitrate staining. The site of 5S rDNA was observed in only one pair of chromosomes, but differing in the marked pair and their location. Based in the differences of the karyotypic formulae and rDNA 5S found between populations on this study and many others available in the literature, it is suggested that this group represents a species complex, and that a new and detailed taxonomical review is necessary.