Migration of amphidromous Rhinogobius species between the mainland and Ryukyu Archipelago just after the opening of the gap was probably not restricted because their larvae can disperse through the coastal areas. With further extension of the gap, their possible migration across the gap gradually decreased; finally, the isolation of populations at either side of the gap was complete. Therefore, we used the opening time of the Tokara Gap as the upper limit of the divergence time between mutually monophyletic groups distributed in the mainland and in the Ryukyu Archipelago. The tMRCA of the mutually monophyletic groups was constrained following a normal prior distribution within the limit of 0–1.7 Myr (mean = 1.55, SD = 0.4; calibration 2, CA2).