The reproducibility, mode of inheritance and polymorphism of amplified fragment length polymorphism (AFLP) markers were examined in ayu Plecoglossus altivelis, (Salmoniformes: Plecoglossidae). The AFLP markers were highly reproducible, their inheritance following Mendelian expectations. The number of fragments amplified (34-134), polymorphic ratio (0.15-0.78) and average heterozygosity (0.02-0.25) of the AFLP markers showed significant variation among six primer pairs and among ayu populations, including a landlocked Lake Biwa population, two amphidromous populations (P. a. altivelis) and two Ryukyu-ayu populations (P. a. ryukyuensis). Although AFLP analysis provided similar results in intra-population diversity and relationships among populations to those found by analyses of allozymes, microsatellites and mitochondrial DNA sequences, AFLPs showed higher polymorphisms and hence greater distinction between genetically close populations. (c) 2005 The Fisheries Society of the British Isles.