Adult forms of members of the Callodistomidae always parasitize the gallbladder of freshwater fishes and occur in Africa and America. This study provides a description of a new South American species belonging in Prosthenhystera from the gallbladder of a characid fish (Bryconamericus ikaa), and ribosomal gene sequences (28S rDNA and ITS1-5.8S-ITS2) are used to demonstrate molecular differences between the new species and congeners as well as explore interrelationships among congeners. Additionally, the first cytological analysis is conducted for a member of the family to determine chromosome number and arrangement. Prosthenhystera gattii n. sp. most closely resembles Prosthenhystera caballeroi in morphology, but the vitellarium is more extensive reaching anterior to the caecal bifurcation in the new species and the uterus is confined to the hindbody in P. gattii n. sp., whereas it extends to the level of the pharynx in P. caballeroi. Also, the testes, cirrus sac, seminal receptacle and the ratio of body length to width are larger in P. gattii n. sp. Independent Bayesian inference analyses of 28S rDNA and ITS1-5.8S-ITS2 sequence fragments produced phylograms that showed P. gattii n. sp. is more similar to Prosthenhystera obesa + Prosthenhystera oonastica than P. caballeroi + two unidentified species of Prosthenhystera, but with poor posterior probability support for the node in the ITS1-5.8S-ITS2-based phylogram. Further, the genetic distance between P. oonastica and P. gattii n. sp. are the largest among Prosthenhystera spp. Cytological analysis revealed ten metacentric chromosomes, which is fewer than the 12–18 chromosomes present in species from the closely related Gorgoderidae.