The genus Hypostomus has a broad geographic distribution in Brazilian rivers and comprises armored catfishes with a very complicated taxonomy due to the absence of morphological autapomorphies. The existence of nearly 10 allopatric populations with different karyotypes suggests that Hypostomusancistroides represents a species complex in the Upper Paraná River basin. In this paper, an unusual karyotype of an isolated H. aff. ancistroides population was investigated. All specimens of this sample have 2n = 66 chromosomes except for 1 male with 2n = 67, most likely due to a supernumerary chromosome. In this population, the sexes are dimorphic, the males are heterogametic, and an XX/XY sex chromosome system is present. Phylogenetic analysis using mitochondrial and nuclear DNAs indicated that this population forms a monophyletic group separate from the other populations of H.ancistroides and may represent an incipient species.