Cytogenetic studies were carried out on seven specimens of Pimelodus heraldoi and sixteen specimens of Pimelodus sp., both from the Parana River basin. The two species had the same diploid number of 56 chromosomes: P. heraldoi with 22M+22SM+6ST+6A and FN of 106 and Pimelodus sp. with 24M+26SM+4ST+2A and FN of 110. NORs were found at the terminal position of the long arm of one pair of ST chromosomes. C-banding (CB) showed in the two species heterochromatin distributed in various chromosomes of the complement, mainly in telomeric regions and in a pair of metacentric chromosomes with strong heterochromatic staining in both telomeres. Treatment only with the fluorochrome CMA3 confirmed in Pimelodus heraldoi and Pimelodus sp. the nucleolar chromosome pair and showed other fluorescent bands. Combined treatment with CB+CMA3 enhanced fluorescent staining of chromosomes in the two fish species evidencing several bands, including in P. heraldoi a chromosome pair showing fluorescent staining in both telomeres.