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Hypostomus is the most specious genus of Hypostominae, composed of several species with high intraspecific morphological and color pattern variation, making their identification a complex issue. One of the species with problematic identification is Hypostomus tietensis that was described from a single specimen, resulting in uncertainties about its color pattern and correct identification. To assist in this context, cytogenetic analyzes were carried out in three putative populations of H. tietensis from the Upper Paraná River basin, one of them from the type locality. The three populations showed considerable cytogenetic differences, with 2n = 72 chromosomes for the population from the type locality and 2n = 76 chromosomes for the others. Terminal NORs were detected (Ag- and 18S rDNA-FISH), being simple for the type locality population (acrocentric pair 23, long arm) and the Pirapó River (subtelocentric pair 11, short arm), and multiple for Do Campo River (subtelocentric pairs 11 and 12, short and long arm, respectively). C-banding was efficient in differentiating the type locality population from the others. Cytogenetic data revealed that populations from Pirapó and Do Campo rivers, although treated until now as Hypostomus aff. tietensis, represent a cryptic species, and those morphological analyses are necessary to differentiate and for describing this new species. [ABSTRACT FROM AUTHOR]