Untargeted metabolomics was employed to systematically reveal the differences in metabolite composition among three parts of Wasabia japonica Matsum (fibrous roots, rhizomes, and leaves). The identified metabolites were further explored using principal component analysis and orthogonal partial least squares-discriminant analysis, followed by KEGG pathway enrichment analysis. In total, 2 883 metabolites were identified in the fibrous roots, rhizomes, and leaves of W. japonica Matsum, among which 1 741 differed significantly. Pairwise comparisons of rhizome vs. fibrous roots, leaves vs. fibrous roots, and rhizome vs. leaves identified 883, 1 213, and 1 108 differential metabolites, respectively. These differential metabolites were found to primarily consist of flavonoids, terpenoids, coumarins, alkaloids, and other functional components, which impacted betaine biosynthesis, flavonoid biosynthesis, tryptophan metabolism, and linoleic acid metabolism significantly. Collectively, these findings demonstrated that the metabolome of W. japonica Matsum varied significantly within its organs, with relevant different biological activities. Therefore, a better understanding of the “part-specific, precise development” of W. japonica Matsum can lay the foundation for the development of new food products.