Abstract Herein we describe two new species of Diapoma, one from the Negro River, a tributary of the Uruguay River in Brazil and Uruguay, and one from the Iguaçu River, in Brazil and Argentina. The new species from the Negro River basin is distinguished from its congeners by the following combination of characters: a black narrow and conspicuous line restricted to the body horizontal septum, incomplete lateral line, tricuspid teeth in the inner series of the premaxilla, and a lower body depth at vertical through the dorsal-fin origin (29.3–32.8% SL in males and 27.7–33.3% SL in females). The new species from the Iguaçu River basin is distinguished from its congeners by the following combination of characters: a discontinuous lateral line, adipose fin hyaline, longer anal-fin base (26.5–32.4% SL), and a longitudinal black stripe along the median region of caudal-fin rays. Additionally, we updated the molecular phylogeny of the genus, including new sequences from these two new species and Diapoma thauma. An identification key for species of Diapoma is presented, modified from previous study.