Freshwater catfishes from the genus Hypostomus have been models for several cytogenetic studies, due to their intense variability in diploid number, chromosome morphology, and the distribution of repetitive DNAs. Taking into consideration the taxonomic complexity inherent to this group, the present study aims to describe the karyotypes of five species of Hypostomus collected in their type localities: Hypostomus albopunctatus (Regan, 1908), Hypostomus hermanni (Ihering, 1905), Hypostomus iheringii (Regan, 1908), and Hypostomus paulinus (Ihering, 1905) from the Piracicaba River (the Upper Paraná River Basin); and Hypostomus mutucae Knaack, 1999 from the Claro River (the Upper Paraguay River Basin). Our results evidenced a great inter-specific diploid-number variation: 2n = 72 (H. hermanni); 2n = 74 (H. albopunctatus); 2n = 76 (H. paulinus); 2n = 80 (H. iheringii); and 2n = 82 (H. mutucae), which reflects the important role of Robertsonian rearrangements in the karyotypic differentiation among these species. The distribution of heterochromatin also varied considerably among species, making it possible to distinguish each analyzed species, as well as to detect microstructural variations among populations of the same species. These data can support taxonomic revisions when further associated with molecular markers and morphological analyses to delimit, more consistently, the taxonomic status of these Hypostomus species, which have a complex taxonomic diagnosis history.