Female ayu Plecoglossus altivelis were sampled from the Mogami River System, Yamagata Prefecture to investigate the occurrence of multiple spawning that has previously been reported in small rivers. Spawning frequencies of the females were estimated by histological observations of the ovary mainly from the degree of degeneration of postovulatory follicles. In the middle and late part of the spawning period, most females showed characteristics of having spawned two or three times, indicating that multiple spawning is a common phenomenon also in a large river. Mean relative batch fecundity (RBF) and condition factor (without gonads) decreased with the number of spawning batches. Size dependency in the occurrence of the multiple spawning and the RBF values was not observed. These results are similar to those reported in previous studies of ayu from a small river near the Mogami River System, and indicate that the occurrence and properties of multiple spawning are almost the same regardless of the river scale. However, it is possible that the ratios of multiply spawned females may differ due to variations in survival rates during the spawning season.