We report the development of nine polymorphic nuclear-encoded microsatellite DNA loci for the African bonytongue (Heterotis niloticus), the only species of the osteoglossiform family Arapaimidae distributed in the Eastern Hemisphere. We describe the process to isolate these loci, and the primers and conditions for amplifying them using the polymerase chain reaction. We tested these primers with a sample of 40 individuals from two natural populations in Benin, West Africa. The number of alleles for the nine microsatellites ranged from 2 to 9, observed heterozygosity from 0.125 to 0.882, and expected heterozygosity from 0.156 to 0.810. No significant deviations from Hardy-Weinberg equilibrium or linkage disequilibrium were observed, and there is no indication of null alleles.