Stock discrimination in the spawning population of ayu, Plecoglossus altivelis altivelis in the Ota River, western Japan was conducted using number of scales above the lateral line and otolith Sr:Ca ratio. Chart pattern in otolith Sr:Ca ratio of hatchery-stocked ayu was different in amphidromous form. Based on chart pattern of otolith Sr:Ca ratio, mixing ratio of hatchery-stocked ayu to the spawning population was estimated at 28%. Number of scales above the lateral line of spawning population marked from 13 to 26 (n=173). Based on number of scales above the lateral line (hatchery-stocked ayu ≦ 17; amphidromous ≧ 18), the mixing ratio of hatchery-stocked ayu was estimated 31%. Consequently, the contribution ratio of hatchery-stocked ayu to the spawning population was around 30% at the Ota River.