Accurately describing biodiversity in tropical regions such as Amazonia is difficult because of insufficient morphological inventories and the lack of studies on the distribution of genetic diversity. Aquatic organisms from Amazonian flooded forests are generally expected to move laterally along the forests during the annual inundation cycle, a behaviour that should promote admixture of populations and reduce within-drainage speciation. We used an unprecedented fine-scale sampling effort and multiple DNA markers to quantify region-wide population differentiation in an Amazonian floodplain forest specialist, the black-wing hatchet fish Carnegiella marthae (Myers, 1927). Our study revealed three previously unsuspected and ancient cryptic species of black-wing hatchet fish in the Rio Negro floodplain (RNF), in central Amazonia. Two species produce occasional first-generation hybrids. The third and rarer species, although found in extreme sympatry with another species, appears to be reproductively isolated, and also differs in external morphology and dentition. Our findings have important implications for guiding conservation management because C. marthae is harvested commercially in the RNF ornamental fishery. They also suggest that the diversity of Amazonian ichthyofauna is vastly underestimated, including that found in landscapes lacking contemporary barriers to account for popula tion divergence and speciation.