A new species of Acanthobothrium in Dasyatis longus from Chamela Bay, Jalisco, Mexico, is a member of a presumed clade of species diagnosed by being anapolytic or nearly so, having more than 100 testes per proglottis, with immature and mature proglottides wider than long to square, aspinose scolex, muscular bothridia fused to the scolex at their posterior ends, H- to V -shaped ovaries, relatively short symmetrical to asymmetrical ovarian arms that extend anteriorly to, or nearly to, the cirrus sac, and vitellaria arranged in fields rather than a single row of follicles. The new species most closely resembles Acanthobothrium terezae from the freshwater stingray Potamotrygon motoro in the following characters: bothridial hooks longer than 200 microns with inner hooks having bent asymmetrical prongs, an average of 130-140 testes per proglottis, and shallow genital atria located posterior to midline of proglottis. The new species differs from A. terezae by having outer hooks approximately the same size and shape as the inner hooks, inner hooks averaging 230 microns rather than 313 microns in total length, and cirrus sacs averaging 255 microns rather than 450 microns in length. The new species is unique among all described species of Acanthobothrium by having a cleft in the posterior margin of each apical bothridial pad. The apparent close relationship of the new species to one inhabiting a Neotropical freshwater stingray provides support for the hypothesized Pacific marine ancestry of Neotropical freshwater stingrays and raises the possibility that the Neotropical freshwater stingrays may not be monophyletic.