A new subspecies of the ayu, Plecoglossus altivelis ryukyuensis, is described on the basis of specimens from Amami-oshima and Okinawa Islands, the Ryukyu Islands, Japan. This new subspecies is distinguished from P. altivelis altivelis by the fewer numbers of pectoral fin rays, longitudinal scales and scales above and below the lateral line, and also by the unique electrophoretic mobilities of several enzymes.