Molecular phylogeny of Potamotrygonocotyle (Monogenea, Monocotylidae) challenges the validity of some of its species. -Zoologica Scripta, 40, 638-658. The marine-derived stingrays Potamotrygonidae are the only chondrichthyans landlocked to freshwaters of Central and South America. The family includes approximately 22 described species organized in four genera widely distributed across the main Atlantic and Caribbean continental drainages. Investigations into the parasite fauna of potamotrygonids have mainly focused on cestodes, with a few studies addressing the biodiversity of monogeneans. Potamotrygonocotyle (Monogenea, Monocotylidae) is composed of 12 species, exclusively found in the gills of species of Potamotrygonidae.This study presents molecular phylogenetic analyses of this group of monogeneans distributed throughout La Plata and Amazonas basins, with the purpose of readdressing the phylogeny of Monocotylidae based on 28S rDNA sequences and of unravelling the phylogeny of its species using data from mitochondrial gene cytochrome c oxidase subunit I and nuclear gene internal transcribed spacer 1. The phylogenetic status of the five tested monocotylid subfamilies and most of their internal relationships are concordant with the results of a previous study, and the monophyletic status of Potamotrygonocotyle based on molecular data is corroborated for the first time. However, the placement of the genus within Monocotylidae is not resolved, as its sister-group relationship with Neoheterocotyle and Troglocephalus is uncertain. Investigations into the relationships within Potamotrygonocotyle support the monophyletic status of nine nominal species and suggest the existence of cryptic lineages for the remaining three. Molecular analyses reveal distinct sister-groups relationships in comparison with a previously published phylogeny for the genus based on morphological data. Finally, the surveys of this study expand the known distribution range of some members of Potamotrygonocotyle.