We investigated four morphologically affiliated species of Macrobrachium in northeastern Asia (M. nipponense and M. formosense, which spawn many small eggs, and M. shokitai and M. asperulum, which spawn a few large eggs). Multi-locus allozyme analysis supported the hypothesis that M. shokitai, endemic to the southernmost island (Iriomote Island) of the Ryukyus, Japan, differentiated recently (approximately 1.0 Myr ago) from nearby M. asperulum populations in Taiwan. M. nipponense and M. formosense are inferred to have differentiated later (0.48 Myr ago). The group of M. shokitai and M. asperulum was presumed to have separated from the group of M. nipponense and M. formosense in several million years, evolving their characteristic reproductive traits. Some populations of M. shokitai and M. asperulum in upland streams show extremely low genetic variability.