SummarySoybean (Glycine max L. Merr.) is a major crop worldwide, providing essential dietary proteins and edible oils for agriculture, food, animal feed, and biofuel industries. However, its limited genetic variation makes it vulnerable to harsh environmental conditions, and its chemical diversity was strongly reduced after long domestication. Wild soybean (Glycine soja Sieb. et Zucc.), the ancestor of the G. max, possesses a high level of genetic diversity and thus present a precious reservoir for improving cultivated soybean. Characterizing metabolomes of different G. soja accessions is crucial in exploring metabolite accumulation patterns for developing metabolic breeding strategies. This study successfully revealed the metabolomes of 64 seed lines of G. soja collected throughout Japan by a widely targeted metabolomics approach, therefore providing a good source of information for further studies to induce the production of bioactive compounds and the defense mechanism to stress.