Ayu Plecoglossus altivelis is one of the most important freshwater fishes in Japan and usually amphidromous. Stock identification was conducted using otolith Sr/Ca analysis for ayu from the Ashida River, Hiroshima Prefecture. In nine of 10 adults sampled from the upper and middle-reaches in August-October 2006, the ratio did not fluctuate and remained at a low level, indicating that these ayu were landlocked fish without staying as larvae and juveniles in coastal marine waters. The landlocked ayu likely reproduce in two artificial lakes (Lake Ashida and Lake Jin-no formed by Hattabara Dam and Mikawa Dam, respectirely) in the Ashida River.