Due to its tectonically uplifted, mountainous landscape and copious rainfall, the Japanese archipelago possesses plentiful, persistent groundwater in alluvial plains. However, Phreatodytes, an endemic water beetle genus found in groundwater, is now critically endangered due to the devastation of subterranean water ecosystems. Phreatodytes beetles were collected from driven wells near the Ooi River estuary in central Japan. The beetles were distinguished from all described species by their thoracic and genital morphologies, and described as a new species, Phreatodytes haibaraensis. The unique habitat of this species is the hyporheic zone of a gravelly riverbed in an estuary, and the habitat represents the eastern limit of the known distribution range of the genus. Molecular phylogenetic analysis based on mitochondrial and nuclear gene sequences revealed that Phreatodytes is a member of the Noteridae and forms a clade with Notomicrus. This suggests that the genus colonized the hyporheic zone from surface waters, and that it adapted to subterranean interstitial life by becoming minute and slender, by losing its eyes, pigmentation, as well as its ability to fly and swim, and by developing a permanent underwater life cycle, and erect hairlike setae on the thorax and elytra. In addition to the beetle, several stygobiontic species, including mollusks and fishes, were found in pumped groundwater. These results suggest that the gravelly riverbed of a rapid river estuary in Central Japan harbors biodiverse, locally differentiated hyporheic organisms comprising a unique subterranean interstitial ecosystem.