The occurrence of gnathiid isopods, a group of parasitic crustaceans on fish, is reported from low-salinity habitats in Miyako-jima Island, Ryukyu Islands, southwestern Japan. In total, 1,096 gnathiid larvae were collected from four of ten surveyed sites, but mostly at two subterranean water sites connected with anchialine waters and/or the sea. Three stages of parasitic zuphea larvae were collected, but no free-living adults. Morphological observations and DNA barcoding show that these larvae are closely related to, and most likely conspecific with, Gnathia limicola a dweller of mud burrows in brackish-water habitats. Our study suggests that G. limicola actively enters into low-salinity subterranean habitats, presumably to search for euryhaline fishes to use as temporary hosts.