Loricariidae (Siluriformes, Hypostominae) is one of the most diverse catfish families. In spite of the wide distribution of loricariids in South America, cytogenetic reports are available for only a few species, mostly from southern and southeastern Brazil. We made the first chromosomal analysis of Hypostomus aff. unae from the Contas River basin in northeastern Brazil. Four populations isolated by short distances but from distinct landscapes were studied based on conventional staining, C-banding, argyrophilic nucleolar organizer regions (Ag-NOR), CMA3/DAPI fluorochrome staining, and fluorescent in situ hybridization with 18S rDNA probes. Although sharing the same diploid number (2n = 76) and NOR locations, each population presented exclusive karyotype formulae and specific patterns of heterochromatic and AT-rich regions. The derived karyotypes of H. aff. unae (2n >54; high number of acrocentrics bearing AT-rich interstitial heterochromatin) indicated a divergent karyoevolution, mostly driven by centric fissions, pericentric inversions and particular heterochromatin dispersion models. This finding of distinct evolutionary units in H. aff. unae will be useful for understanding the natural history of loricariids from relatively unexplored coastal basins in South America.