Catfishes of the genus Loricariichthys are widely distributed in the Platina Basin. Considering that the cytogenetic knowledge of Loricariichythys is underestimated, this study assessed Loricariichthys anus and Loricariichthys platymetopon through different chromosome bandings, to define the mechanisms determining the variability in these species. Cytogenetic analyses evidenced a high degree of similarity in relation to the 2n (54 chromosomes), as well as to the distribution of heterochromatin. Despite the apparent conservatism, it was possible to differentiate between these species, especially in relation to the location of the 18S rDNA genes. An interpopulation variation in the karyotype formula was detected only in L. anus, showing the existence of different karyotypes, probably due to the geographical isolation between Laguna dos Patos and Tramandaí River. The maintenance of the 2n=54, along with the different karyotypes observed in L. anus, the differential nucleolus organizer regions position, as well as the sexual chromosome system ZZ/ZW in L. platymetopon, makes the participation of pericentric inversions in the karyotypic evolution of these species evident. These structural rearrangements were important for chromosome evolution of these two species, because they probably promoted the postzygotic barriers to reproduction, significantly contributing to the speciation process between them.