© 2014, The Ichthyological Society of Japan. The distribution of fluvial fish was surveyed at 55 sites on Yakushima Island, Japan, which has precipitous mountains and waterfalls (below- or no-waterfall sites: 31; above-waterfall sites: 24). Eleven diadromous and one river resident (introduced Oncorhynchus masou masou) species were found, but absolutely no fish were detected at the 18 above-waterfall sites. Statistical analyses revealed that the presence of waterfalls (> 5 m in height) below the sites had a significant negative effect on fish distribution, suggesting that waterfalls prevent migration of diadromous fishes. We found Rhinogobius brunneus populations above high waterfalls such as Nunobiki Falls (50 m) and Ohko Falls (88 m). Otolith Sr:Ca ratios and mitochondrial DNA (cyt-b region) were examined to determine the migratory history and genetic properties of these populations; the Sr:Ca ratios indicated that the populations had a landlocked life cycle, whereas the genetic endemism/isolation of landlocked populations was unexpectedly absent. There is no clear explanation for this phenomenon, but fairly infrequent individuals of nonlandlocked type might have migrated beyond the waterfalls with their sucker-like organ and mated with landlocked populations, disrupting the genetic isolation of landlocked populations.