Megalonaias is the most geographically widespread genus of the subfamily Ambleminae and is distributed across much of the eastern half of North America, from Minnesota to Nicaragua. Despite the large geographic distribution, the species-level diversity of Megalonaias is quite depauperate (2 spp.), suggesting the genus may not be constrained by the same physical, ecological, or physiological barriers that limit dispersal in many other amblemines. However, this hypothesis is contingent on the assumption that the current taxonomy of Megalonaias accurately reflects its evolutionary history, which remains incompletely understood due to the marginalization of Mesoamerican populations in systematic research. Using one mitochondrial (COI) and one nuclear marker (ITS1) sequenced from 41 individuals distributed across both the Nearctic and Mesoamerican ecoregions, we set out to better understand the species boundaries and genetic diversity within Megalonaias. The reconstructed molecular phylogeny and the observed genetic diversity suggests that Megalonaias is a monotypic genus and that Megalonaias nickliniana, currently considered a federally endangered species, is not a valid species. These results are discussed in the context of their systematic and conservation implications, as well as how the unusual life history strategy of Megalonaias may be influencing its molecular diversity.