We conducted a review of the Neotropical genus Acrobrycon. A previous study synonymized the two recognized species in the genus: A. ipanquianus, distributed from the western portion of the Amazon River to the northwest region of the La Plata River Basin, and A. tarijae, described from the Lipeo River in Bolivia. We revisited this result by collecting additional morphometric, meristic, and genetic data (COI mitochondrial gene) for 24 individuals distributed along La Plata River Basin in Argentina, and discussed our results in the context of multiple biogeographic process of isolation in this basin. Our results revealed a more complex history of diversification and geographic distribution across Acrobrycon species than previously suspected. We present new evidence that leads us to reconsider the validity of A. tarijae, which is distinguished from A. ipanquianus by the number of vertebrae (37-39 vs. 41-42), pleural ribs (12-13 vs 14), and unbranched anal-fin rays (v-vi vs. ii-iii). These results were supported by molecular analysis which showed a genetic divergence >4% between both nominal species, also, indicated two major genetic clusters of A. tarijae: the first group consisted of specimens from the Bermejo, Pilcomayo, Itiyuro and Juramento River Basins (northern Argentina), and the second group is composed by specimens from the southernmost basins, such as the Salí River in Tucumán and the Quinto River in the province of San Luis. These results suggest that the genetic structure may be a consequence of the geographical distance that separates the two groups and the type of drainage.