Abstract Cytogenetic analyses were performed on Hoplias malabaricus (Bloch, 1794) from the Taquari River, Parana River drainage, revealing two sympatric karyomorphs. One karyomorph was characterized by 2n = 40 m/sm and 2n = 39 m/sm chromosomes for females and males, respectively, and an X1X1X2X2/X1X2Y sex chromosome system. In the second karyomorph, specimens showed 2n = 42 m/sm chromosomes, without sex-related heteromorphism. Both karyomorphs were characterized by a distribution of heterochromatin in the pericentromeric and telomeric regions. In addition to the differences in the diploid numbers and the sex chromosomes, the GC-rich sites and the nucleolar organizer regions also showed clear differences between the karyomorphs. Coupled with the occurrence of unique chromosomal features within each karyomorph, the fact that hybrids have not been identified in the sampled population provides additional support of the existence of a species complex in H. malabaricus.