The duration of spawning markers (e.g. signs of previous or imminent spawnings) is essential information for estimating spawning frequency of fish. In this study, the effect of temperature on the duration of spawning markers (i.e., oocytes at early migratory nucleus, late migratory nucleus, and hydrated stages, as well as new postovulatory follicles) of an indeterminate multiple-batch spawner, Japanese f lounder (Paralichthys olivaceus), was evaluated. Cannulation was performed to remove samples of oocytes, eggs, and postovulatory follicles in individual females at 2–4 hour intervals over 27–48 hours. The duration of spawning markers was successfully evaluated in 14 trials ranging between 9.2° and 22.6°C for six females (total length 484–730 mm). The durations of spawning markers decreased exponentially with temperature and were seen to decrease by a factor of 0.16, 0.36, 0.30, and 0.31 as temperature increased by 10°C for oocytes at early migratory nucleus, late migratory nucleus, and hydrated stages, and new postovulatory follicles, respectively. Thus, temperature should be considered when estimating spawning frequency from these spawning markers, especially for those fish that do not spawn synchronously in the population.