Fifty-six species or approximately 20% of the Neotropical characiform superfamily Anostomoidea (families Anostomidae, Chilodontidae, Curimatidae and Prochilodontidae) occur in the freshwaters of French Guiana, Guyana, and Suriname although these regions span only approximately 6% of the native range of this morphologically and ecologically diverse superfamily. As a result of the high diversity of anostomoids in the Guianas, the taxonomic and/or geographic boundaries of many species are poorly understood, particularly within the family Anostomidae. This contribution unites results of prior taxonomic revisions with surveys of the holdings of the superfamily in seven major natural history collections to provide distribution maps and keys to the identification of all anostomoid species known to occur in the Guianas. These maps reveal six major areas of endemism: the Takutu and Ireng rivers (Rio Branco drainage, Amazon basin), the Barima and Waini rivers (Orinoco drainage), the Essequibo-Rupununi River system (including the Demerara and Berbice rivers), a union of the Corantijn, Nickerie, Coppename, Saramacca and Suriname river drainages, the Marowijne/Maroni River system (including the Litani and Mana rivers), and the Oyapock-Approuague River basins. Of these, the Essequibo-Rupununi system harbours the most exceptionally diverse assemblage of anostomoid species, likely as a result of faunal exchange between the Rio Branco and Essequibo River across the Rupununi portal.