Abstract We herein document intromittent organs in four Peruvian populations of inseminating fishes of the family Characidae, genus Monotocheirodon, which may comprise new species. All females histologically analyzed from these populations, as well as a female of Monotocheirodon pearsoni, had spermatozoa within the ovary, confirming insemination. Male M. pearsoni produced aquasperm with spherical nuclei (diameter 1.6 μm), with no intromittent organ. On the other hand, males from the Peruvian populations of Monotocheirodon produced spermatozoa with slightly ovoid to elongate nuclei (lengths 1.8–4.1 μm) and possessed distinct intromittent organs. Some populations with more elongate sperm nuclei had longer intromittent organs. Each intromittent organ consisted of an elongate extension of the body wall encompassing an extension of the common sperm duct and urinary duct, which joined with one another near the base of the organ and opened to the outside at the pointed tip of the organ. Skeletal muscle cells are...