Dioscorea opposita Thunb., a traditional medicinal and food homologous plant, has been used for thousands of years in folk medicine and nutritious food, partially due to the metabolites. However, detailed information on the metabolites in Dioscorea opposita Thunb. peel is still scarce. Herein, an ultra-high-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry-based metabolomics approach was used to reveal the comprehensive metabolites in Dioscorea opposita Thunb. peel. The proposed method provided tentative identification of 307 metabolites. Among them, 286 metabolites are reported herein in Dioscorea opposita Thunb. peel for the first time, namely: 87 lipids, 49 phenolic acids, 34 flavonoids, 29 amino acids and derivatives, 28 organic acids, 14 alkaloids, 10 lignans, and coumarins, eight saccharides and vitamins, six nucleotides and derivatives, two quinones, and 19 others. The obtained results highlight the importance of Dioscorea opposita Thunb. peel as a promising source of bioactive ingredients, and form a basis for future quantitative and bioavailability studies. © 2023 Wiley-VCH GmbH.