Spatiotemporal features of anthocyanin accumulation in a model legumeLotus japonicus(Regel) K.Larsen were elucidated to develop criteria for the genetic analysis of flavonoid biosynthesis. Artificial mutants and wild accessions, with lower anthocyanin accumulation in the stem than the standard wild type (B-129 ‘Gifu’), were obtained by ethyl methanesulfonate (EMS) mutagenesis and from a collection of wild-grown variants, respectively. The loci responsible for the green stem of the mutants were named asVIRIDICAULIS(VIC). Genetic and chemical analysis identified two loci, namely,VIC1andVIC2, required for the production of both anthocyanins and proanthocyanidins (condensed tannins), and two loci, namely,VIC3andVIC4, required for the steps specific to anthocyanin biosynthesis. A mutation inVIC5significantly reduced the anthocyanin accumulation. These mutants will serve as a useful system for examining the effects of anthocyanins and proanthocyanidins on the interactions with herbivorous pests, pathogenic microorganisms and nitrogen-fixing symbiotic bacteria,Mesorhizobium loti.