A collection of 94 F(6) individuals derived from crosses between Lotus japonicus, Gifu B-129 (G) and Miyakojima MG-20 (M) were used for mapping. By using the HEGS running system, 427 EcoRI/MseI primer pairs were selected to generate a total of 2053 markers, consisting of 739 G-associated dominant markers, 674 M-associated dominant markers, 640 co-dominant markers, 95 SSR markers and 2 dCAPS markers. Excluding heavily distorted markers, 1588 were mapped to six chromosomes of the L. japonicus genome based on the 97 reference markers. This linkage map consisted of 1023 unique markers (excluding duplicated markers) and covered a total of 508.5 cM of the genome with an average chromosome length of 84.7 cM and interval distance of 0.50 cM. Fifteen quantitative traits loci for eight morphological traits were also mapped. This linkage map will provide a useful framework for physical map construction in L. japonicus in the near future.