An environmental DNA detection method is utilized for revealing the truly cryptic species diversity of the subterranean amphipod genus Pseudocrangonyx in the Japanese Archipelago. Water samples were obtained from groundwater in Kyoto and Tokushima, where are the type localities of P. kyotonis and P. shikokunis, respectively. One litter each of 42 water samples in Kyoto and of 19 water samples in Tokushima was collected. Mitochondrial 16S rRNA region was amplified from eDNA samples: a two-step polymerase chain reaction protocol was applied for the library preparation. Environmental DNA method confirmed occurrences of Pseudocrangonyx amphipods in Kyoto for the first time more than 90 years since the previous recorded detection.