Abstract <p>The extensive number of <i>Hypostomus</i> species necessitates further research into the cytogenetics of this group. This study conducted a crucial comparative cytogenetic analysis in two populations of <i>H. nigromaculatus</i> with the aim of identifying potential interpopulation chromosomal markers and contributing significantly to our understanding of the evolutionary trends of this species. Both populations of <i>H. nigromaculatus</i> exhibited a consistent diploid chromosome number of 2n = 76, along with distinct karyotypes and fundamental number values. A single 5S ribosomal DNA site and multiple nucleolus organizer regions sites were detected. Additionally, the presence of heterochromatin in chromosomes carrying nucleolus organizer regions was reported, except for individuals of <i>H. nigromaculatus</i> from the Bom Jesus Stream. Pericentric inversions (or other types of centromeric shift) and/or deletions were found to be essential in the karyotype evolution and differentiation among <i>H. nigromaculatus</i>. These findings are valuable for enhancing our understanding of karyotypic evolution in <i>Hypostomus</i>.</p>