Hemisorubim platyrhynchos is a medium- to large-sized pimelodid catfish distributed along several river basins of the Neotropical Region, noteworthy for representing an important fishery source. In this work, Hemisorubim platyrhynchos from three isolated populations were cytogenetically analyzed. The karyotype shows a diploid number of 2n=56 chromosomes comprising 22m, 16sm, 10st, 8a (FN=104). NORs detected by AgNO3 were located in the terminal regions of the short arm of a st chromosome pair, as confirmed by CMA3 and FISH using an 18S rDNA probe. C-banding revealed a small amount of heterochromatin in chromosomes, including the NORs, and one biarmed pair that showed conspicuous positive bands on both arms. This fact was also evidenced when using other banding techniques, such as RE (AluI), and indicates that this pair constitutes a species-specific cytogenetic marker.