The African bonytongue (Heterotis niloticus), one of two living species of the primitive teleost family Arapaimidae, constitutes an important artisanal and commercial fishery in West Africa. This species has also been proposed for wide aquaculture use in Africa. Despite its importance, information on the levels of genetic differentiation for this fish in Benin is lacking, which can contribute to its conservation and management. In this study, regional and local scale genetic differentiation of the African bonytongue in Benin, West Africa, was examined using six microsatellite markers. In total, 221 H. niloticus individuals were sampled from 12 localities in Benin that include three river basins: Oueme-So (ten localities sampled); Mono (one locality); and Niger (one locality). The results showed a high degree of genetic differentiation between African bonytongue samples from the three river basins, which was expected given the barriers for dispersal for aquatic organisms. For the Oueme-So floodplain, they indicate high and homogeneous gene flow, suggesting that seasonal flooding facilitates gene flow across this region. The information obtained from this study will be useful for defining management units for H. niloticus in Benin, and caution against the translocation of individuals from different basins.