Changes in the proportions of river‐ and lake‐produced eggs of a landlocked amphidromous fish, ayu (Plecoglossus altivelis altivelis) in the Lake Biwa water system, Japan, were monitored by stable isotope analysis, based on different δ15N and δ13C values of prey organisms between the lake and its tributaries. During the 3 month reproduction season, the δ15N values of spawned eggs decreased with time. This result implies that there was a shift from lake‐produced eggs to river‐produced eggs within a reproductive season, based on the observation that adult fish in the lake had previously been shown to have eggs with distinctly higher δ15N values in their ovaries than those in the tributaries. This explanation was also supported by the change in δ13C values of the spawned eggs. Furthermore, eggs with lower δ15N and higher δ13C values tended to be spawned at less variable depths, suggesting that females spawning river‐produced eggs selected the spawning sites from a narrower range. We conclude that stable isotope ratios of spawned eggs can be indicators of the relative contributions of different food chains and can enable comparisons of reproductive characteristics between types of egg.