Prosthenhystera oonastica n. sp. is described as a cryptic species from the gall bladder of three ictalurid catfishes, Ictalurus punctatus (Rafinesque), Ictalurus furcatus (Valenciennes), and Pylodictis olivaris (Rafinesque), in rivers in the southeastern United States. The species was originally named by Wilmer A. Rogers in 1979 but never formally described. Material used for the description consists of two specimens of Roger's original material and ten new specimens. We found no significant morphological features that are useful for discriminating between the new species and its closest relative Prosthenhystera obesa (Diesing, 1850) Travassos, 1922 that occurs in the gall bladders of freshwater characiform, perciform and siluriform fishes, ranging from South America to southern Mexico. However, we found substantial differences in the large subunit ribosomal DNA (partial 28S rRNA gene) between the two species justifying the naming of the new species. Prosthenhystera oonastica n. sp. is readily differentiated from Prosthenhystera caballeroi Jiménez-Guzmán, 1973 that occurs in the gall bladders of characid fishes in Central America and Mexico, by having a relatively straight or bent rather than highly convoluted oesophagus, a relatively smaller ovary, smaller and less coalesced vitelline follicles, narrower caeca and smaller eggs. Comparison of ribosomal DNA (partial ITS1, 5.8S, ITS2, and partial 28S gene) between P. oonastica n. sp. and P. caballeroi revealed large differences between the two species. Phylogenetic analysis based on partial 28S rRNA gene sequences from the three studied species of Prosthenhystera Travassos, 1922 and related digenean taxa revealed a closer relationship between P. oonastica n. sp. and P. obesa than either has had with P. caballeroi.