Lotus japonicus has attracted attention as a model plant legume for molecular genetic research, and several mutants defective in nodulation and mycorrhizal symbiosis have been developed from the standard accession Gifu B-129. However, as a model system, Gifu has long lacked an appropriate crossing partner for use in various genetic analyses. In a search for an appropriate partner for Gifu, we have collected plants from 15 localities throughout Japan, and analyzed their levels of DNA polymorphism (also in comparison to the African species L. filicaulis) by AFLP (Amplified Fragment Length Polymorphism) combined with the use of a high-throughput electrophoretic screening system termed HEGS (High-efficiency genome scanning) developed by us, using 31 primer pairs. Plants of the accession Miyakojima MG-20 showed the highest level of polymorphism relative to Gifu (over 4%). When HEGS is used for screening, this level is sufficient to permit systematic positional cloning of mutant genes. Segregation in the F2 of the Gifu-derived symbiotic mutations Ljsym70, Ljsym72, Ljsym74-1 (alb1-1) and Ljsym78-1 from a cross with Miyakojima was normal, while the ratios seen from a cross with L. filicaulis were distorted. Miyakojima displays several traits that distinguish it from other Japanese accessions: low concentrations of anthocyanin in the stem and petals, few trichomes, a more upright habit, broad leaflets and petals, and large black seeds. The first two traits, which are controlled by single recessive genes, serve as useful markers for following mutant crosses.