Abstract The family Potamotrygonidae is part of the Neotropical ichthyofauna and has the largest diversity of freshwater elasmobranchs in the world. The neurocranium in Potamotrygonidae is an elongated and dorsoventrally flattened structure that articulates anterolaterally with the anterior portion of the propterygium, posteriorly with the sinarcual, and posterolaterally with the hyomandibula. This anatomical structure is comparatively described in Potamotrygonidae in an effort to reveal new phylogenetically relevant characters. Additionally, a new genus-level phylogeny of the family is proposed herein based on 60 novel morphological characters. The subfamily Potamotrygoninae, whose monophyly is well sustained by previous studies, is further supported herein by a variable number of cranial synapomorphies (up to 17), depending on the optimization adopted. Furthermore, we also recovered Potamotrygon and Plesiotrygon as consecutive sister-groups to a clade including Heliotrygon and Paratrygon. The neurocranium of potamotrygonids and its anatomical variation is also discussed in the context of previous morphological and molecular phylogenies of Neotropical freshwater stingrays.