Stock separation for spawning population of ayu Plecoglossus altivelis in the Gouno River was conducted using otolith Sr/Ca concentration ratio. Based on stock enhancement record, coexistence of amphidromus, hatchery-reared and landlocked ayu can be expected in the spawning population. In case of stock separation in the spawning population carrying out the otolith Sr/Ca ratio, 29 and 13 out of 42 individuals were categorized as the hatchery-reared and amphidromus ayu, respectively. The result of no landlocked ayu in the spawning population suggests that the contribution of the landlocked ayu to spawning and reproduction can be neglected at the Gouno River.