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Despite its relevance for ecology, evolution and conservation of species, natural hybridization and hybrids biology are still poorly studied in freshwater fish. Here, we tested the hypothesis that sympatric species Astyanax paranae and A. fasciatus are able to interbreed in the natural environment and presented evidence for the first record of hybridization between these species. We analyzed anatomical traits, gametogenesis, reproductive biology, and genetic variations of the COI and S7 genes of both species and putative hybrids. Intermediate morphometric and meristic features were observed in hybrids when compared to A. paranae and A. fasciatus. Overlap in reproductive season was showed for these species, with greater reproductive activity from August to January, but hybrids did not present any sign of gonadal maturation. Oogonia and perinucleolar follicles as well as spermatogonia and primary spermatocytes were found in hybrids, but previtellogenic and vitellogenic follicles, spermatids, and spermatozoa were absent. Moreover, several alterations in gametogenesis were detected, such as interrupted meiosis in both males and females, vacuolated and degenerated germ cells, increased interstitial tissue, and presence of immune cells. Molecular analyses supported the hypothesis of hybridization between A. paranae and A. fasciatus. Overall, our multidisciplinary approach also provides strong evidence that hybrids are infertile.