The Neotropical fish Heptapterus mustelinus, collected in the Pindorama stream of the upper Paraná River basin (Brazil), was studied cytogenetically, verifying 54 chromosomes (26m + 18m + 4st + 6a). This diploid number has not been reported
among the Heptapteridae that have been studied to date. Unlike most species of the family, there were multiple Ag‐nucleolar organizer regions (NOR) and heterochromatin present in the centromeric region of most of the chromosomes of the complement, being Chromomycin A3 (CMA3+)/4′,6‐diamidino‐2‐phenylindole
(DAPI−) in nine chromosomal pairs, besides the one that coincides with the Ag‐NORs. The data presented in this work reveal a different path in the karyotypic evolution of H. mustelinus when compared to the others Heptapteridae genera.