The blackwing hatchetfish, Carnegiella marthae, is a small characin species distributed in forest streams of the Negro and upper Orinoco River basins in Amazonia. Freshwater hatchetfish are popular in the aquarium trade and represent an economic resource for the riverine people from middle Rio Negro, in Brazil. We isolated and characterized seven microsatellite DNA loci for the blackwing hatchetfish. Number of alleles and heterozygosity per locus in a sample of 30 fish ranged from three to 17 and from 0.19 to 0.87, respectively. These microsatellite loci provide powerful markers for studies on taxonomy, management and phylogeographic history of Amazonian hatchetfish.