We explored a method to accurately and rapidly assess the species diversity of riverine benthic fauna by DNA metabarcoding utilizing next-generation sequencing, and confirmed that DNA metabarcoding can be used to assess the relative abundance and wet weight of each species. We tested the hypothesis that environmental DNA of a variety of species is enriched in the riverbed at the upstream end of a gravel bar where river water is submerged, and found no significant differences in species diversity in the porewater at the upstream end of the gravel bar, the downstream end, and the intermediate sites. The species detected at these three sites also differed significantly.