Stamen from Paeonia ostii 'Fengdan Bai' and Paeonia rockii is rich in phenolic compounds and popularly used as tea materials with various pharmaceutical functions. In order to investigate whether stamen from other tree peony cultivars could be used as a natural antioxidant, the quality of stamen from thirty-five cultivars collected from the same garden was evaluated based on their phenolic composition and content by high-performance liquid chromatography analysis and in vitro antioxidant properties coupled with comprehensive chemometrics analysis. The results revealed that phenolic contents and antioxidant capacities of tree peony stamen were unique and cultivar dependent. Stamen from 'Zi Erqiao' exhibited the highest total phenolic and flavonol content, and strongest antioxidant activities, while that of 'Fengdan Bai' and P. rockii were at below-average levels among test samples. Further, thirty-seven cultivars of tree peony were divided into three major groups with a significant difference in total metabolites content and antioxidant properties, which were mainly contributed by six phytochemical compounds. Among these, naringin and benzoylpaeoniflorin were found to be critical chemical markers for the identification of tree peony stamen with high quality by chemometric analysis. Moreover, correlation analysis suggested that stamen from the earlier flowering cultivars with the hidden pistil, double petal, shorter thrum, more carpel and volume were possibly of higher quality. Together, cultivars with stamen enriched in phenolics and antioxidants properties, and their relevant critical phenotypic and phytochemical traits were screened out. This study would benefit the rapid identification of tree peony stamen with high quality and provide a valuable reference for its development and utilization as functional foods and pharmaceutical resources.