Dendrorchis retrobiloba Volonterio & Ponce de León, 2005 was first described from the swim bladder of Astyanax fasciatus (Cuvier, 1819) from Montevideo, Uruguay. In this study, we necropsied specimens of A. aff. fasciatus collected from Lake Guaíba, in Rio Grande do Sul, Brazil, for the analysis of their helminths. The digeneans were identified as D. retrobiloba by their elongated body, oral sucker longer than ventral sucker, and 2 posterior lobes. This is the first report of D. retrobiloba in A. aff. fasciatus from Lake Guaíba and extends the known geographic distribution of this parasite.