Wheatgrass is recognized for its nutritional and medicinal properties, partly attributed to its flavonoid content. The objective of this study was to assess the flavonoid content and antioxidant properties of wheatgrass obtained from a wide range of 145 wheat cultivars, which included Chinese landraces (CL), modern Chinese cultivars (MCC), and introduced modern cultivars (IMC). The flavonoids were extracted using a solution of 80% methanol, and their content was evaluated using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). The results revealed the assessed cultivars showed significant variation in their total flavonoid content (TFC), with MCCs generally having higher amounts compared to CLs. PCA analysis demonstrated clear variations in flavonoid profiles between different cultivar groups, emphasizing the evolutionary inconsistencies in wheat breeding. The antioxidant assays, ABTS, DPPH, and FRAP, exhibited robust abilities for eliminating radicals, which were found to be directly associated with the amounts of flavonoids. In addition, this study investigated the correlation between the content of flavonoids and the ability to resist powdery mildew in a collection of mutated wheat plants. Mutants exhibiting heightened flavonoid accumulation demonstrated a decreased severity of powdery mildew, suggesting that flavonoids play a protective role against fungal infections. The results highlight the potential of wheatgrass as a valuable source of flavonoids that have antioxidant and protective effects. This potential is influenced by the genetic diversity and breeding history of wheatgrass. Gaining insight into these connections can guide future wheat breeding endeavors aimed at improving nutritional value and in strengthening disease resistance. The current finding provides critical information for developing wheatgrass with high flavonoid content and antioxidant activity.