Abstract: Wheat powdery mildew (Bgt), a fungal seeding disease, is a threat to wheat agriculture worldwide. In this study, iron shell wheat (Triticum aestivum ssp. yunnanense King) was used as the research material. Extensive targeted metabolomic analysis was conducted on the leaves of the susceptible strain iron shell wheat 1 (DiKe 1) and the normal resistant strain Zhenkang iron shell wheat (Yunmai 0030). Furthermore, the dynamic physiological changes in metabolite levels between the two strains were assessed. Significant differences were identified in tryptophan and pyrimidine metabolism as well as isoquinoline alkaloid biosynthesis. The main metabolites were phenolic acids, flavonoid carboglycosides, indole alkaloids, phenolic amines, amino acids, and their derivatives. The defense response to powdery mildew in the iron shell wheat was dominated by tryptophan metabolism. The importance of L-dopa and tyramine in powdery mildew resistance provides new insights into the relevant mechanisms and can help identify antigens for use in the breeding of powdery-mildew-resistant wheat varieties. © 2023, Pleiades Publishing, Ltd.