We studied the chromosomes of Rhamdia quelen. All specimens presented 2n = 58 and 36m+16sm+6st and FN = 116 and showed metacentric B chromosomes of medium size. The specimens from Água dos Patos river, Água das Pedras river and fish farm station showed a secondary constriction in the short arm of pair 28 (st), coincident with the NOR, while in the Taquari river population, the NOR was seen in the terminal region of the short arm in pair 23 (sm), where the homologous chromosomes showed heteromorphism for this region. The NORs were coincident with C-positive band and showed bright fluorescent signals when stained with CMA3. FISH using an 18S rDNA probe was performed in the fish farm station specimens, confirming one pair of chromosomes with the ribosomal cistrons. The pattern of heterochromatin distribution was similar in all populations, being limited to weak staining in the terminal regions of the NOR bearing chromosomes. The specimens of R. quelen from a fish farm station were karyotypically similar to those from the natural environment.