Examination of the spiral intestines of 44 freshwater stingrays, Potamotrygon motoro, from tributary rivers of the Paraná River in Argentina, allowed for the collection of specimens of an undescribed species of Acanthobothrium. Acanthobothrium ramiroi n. sp. can be distinguished from all other congeners by the combination of the following characters: asymmetrical hooks (medial and lateral hooks conspicuously different in size and form, with axial prong of medial hooks stouter than abaxial prong), hook size (total length of medial hooks up to 242 μm, total length of lateral hooks up to 239 μm), bothridia not fused to the scolex proper at posterior ends, worm size (51–84 mm long), and the presence of a conspicuous vaginal sphincter. The new species is different from all other species of Acanthobothrium in freshwater potamotrygonids, except Acanthobothrium terezae, in having conspicuous asymmetrical hooks. The main differences that allow for the distinction between A. ramiroi and A. terezae include hook size, the way the bothridia are attached to the scolex proper, and the shape of the older gravid segments. The discovery of a new species of Acanthobothrium from a potamotrygonid extends our understanding of the diversity of the genus in freshwater stingrays in South America.