Two new genera and species of freshwater turtle blood flukes (TBFs) are described herein based on specimens infecting the nephritic and mesenteric blood vessels of “matamatas” (a side-necked turtle, Chelus fimbriata [Schneider, 1783] [Pleurodira: Chelidae]) from the Amazon River Basin, Peru. These taxa comprise the first-named species and the first-proposed genera of freshwater TBFs from the continent of South America. A new comparison of all TBF genera produced 6 morphologically diagnosed groups that are discussed in light of previous TBF classification schemes and a novel phylogenetic hypothesis based on the nuclear large subunit ribosomal DNA (28S). Considering external and internal anatomical features, species of the new genera (Atamatam Bullard and Roberts n. gen., Paratamatam Bullard and Roberts n. gen.) are most similar to each other and are together most similar to those of several marine TBF genera. The 28S phylogenetic analysis supported the monophyly of all 6 morphologically diagnosed groups of genera. Most notably, the freshwater TBFs of South America comprise a derived group nested within the clade that includes the paraphyletic marine TBFs. Not surprisingly in light of morphology, another marine TBF lineage (Neospirorchis Price, 1934) clustered with the freshwater TBFs of Baracktrema Roberts, Platt, and Bullard, 2016 and Unicaecum Stunkard, 1925. Our results, including an ancestral state reconstruction, indicated that (1) freshwater TBFs have colonized marine turtles twice independently and that (2) the South American freshwater TBFs comprise a marine-derived lineage. This is the first evidence that TBFs have twice independently transitioned from a marine to freshwater definitive host. Marine incursion is considered as a possible mechanism affecting the natural history of marine-derived freshwater TBFs in South America. A dichotomous key to accepted TBF genera is provided.