Prosorhynchoides extenuatus n. sp. is described based on adult worms collected from the intestine of the chain pickerel, Esox niger Lesueur, 1818, from the Pascagoula River, Jackson County, Mississippi, U.S.A. Sequences of the ribosomal DNA from adult specimens (internal transcribed spacer region 2, and partial 28S ribosomal DNA gene) were identical to those of a cercaria developing in branched sporocysts within the Gulf wedge clam, Rangia cuneata (G. B. Sowerby I, 1832) and a metacercaria we collected from the threadfin shad, Dorosoma petenense (Günther, 1867) in the same river. The cercaria conforms to the description of bucephalid cercaria C Wardle, 1990, that was originally described from the same host in Texas, U.S.A. Novel sequence data are also provided for Prosorhynchoides potamoensis Curran & Overstreet, 2009, and Prosorhynchoides pusillus (Stafford, 1904) Margolis & Arthur, 1979. The 28S rDNA fragments from these species are aligned with bucephalid sequences available in GenBank and their relationships are analyzed using Bayesian inference analysis. The new species formed a branch within a clade of marine bucephalines, whereas P. potamoensis and P. pusillus branched within a clade of North American freshwater species currently classified in Bucephalinae and Paurorhynchinae. This study is the first to employ nucleotide-based evidence to demonstrate a 3-host life cycle from a North American bucephalid.