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Abstract Background Hypostominae is a subfamily of the family Loricariidae that has a great diversity of species. Accordingly, testicular studies in fish can contribute to the phylogeny and taxonomy of species and to the comparison of reproductive aspects between species. Thus, this work aimed to characterize the testicular morphology and spermatogenesis of the Hypostominae species Baryancistrus xanthellus, Peckoltia oligospila and Hypancistrus zebra. Results B. xanthellus, P. oligospila and H. zebra had an anastomosed tubular type of testis. The germinal epithelium was continuous with unrestricted spermatogonia, and the proliferative, meiotic and spermiogenic phases were defined in all species. In the spermiogenic phase, spermatids were classified as initial, intermediate and final. Only in B. xanthellus in the final phase was there nuclear rotation. The sperm for the three species had a head with an oval shape and a single flagellum composed of the short midpiece, principal piece and end piece. B. xanthellus and P. oligospila showed a cylindrical flagellum and H. zebra showed projections that produced a flattened appearance. Conclusions On the basis testicular structure and ultrastructural characteristics of the germ cells, there was a greater relationship between B. xanthelus and P. oligospila, while H. zebra had particular characteristics. These aspects show that despite belonging to the same subfamily, the species have distinct biological characteristics.