Steindachneridion melanodermatum is the largest catfish of the Lower Iguaçu River and is endangered due to the habitat fragmentation caused by dams. Currently, the wild population’s last refuge is restricted to an area of 190 km. This study presents the first analysis of its genetic diversity and population structure, using microsatellite loci and mtDNA. The population has an adequate level of genetic diversity, but signs of a recent bottleneck were observed. The Baixo Iguaçu Hydroelectric Power Plant has recently fragmented the population and threatened it with extinction in a reduced area of nearly 30 km. Based on our results, we strongly advise against the stocking of breeding specimens below the Salto Caxias HPP to not compromise the integrity of the native gene pools at the receptor sites. In addition, we recommend manual fish transposition, trap-and-haul, to maintain the genetic connectivity of individuals upstream and downstream of the dam as a conservation strategy. Furthermore, studies on behavior and swimming capacities, and suitable fishways for this species must be developed. We strongly recommend that the Lower Iguaçu River and its tributaries be protected and preserved as free from additional barriers to prevent future habitat disruption for the benefit of S. melanodermatum and several other endemic and endangered species.