A cytogenetic study was conducted on two species of the genus Pimelodus that were collected from the Piquiri river, Paran?, Brazil: Pimelodus paranaensis and Pimelodus heraldoi. Both had a diploid number of 2n=56 chromosomes and a fundamental number (FN) of 104. In P. paranaensis, the karyotype consisted of 22m+22sm+4st+8a chromosomes, whereas the karyotype of P. heraldoi consisted of 18m+24sm+6st+8a. The AgNORs were localized in the terminal region of the long arm in one pair of subtelocentric chromosomes, pair 24 in P. paranaensis and pair 23 in P. heraldoi. The latter species showed size heteromorphism of these regions between the chromosome homologues. Heterochromatin was distributed mainly in the terminal regions in the two species. CMA3-positive staining was observed in some chromosomes, besides being associated with NORs, which were all DAPI-negative, in both species of Pimelodus. C-banding plus CMA3 and DAPI showed that most of the heterochromatic regions were rich in AT bases in P. paranaensis and P. heraldoi.