As recorded in “Chinese Pharmacopoeia”, lotus plumule can be used as medicine. In present study, flavonoids in plumules of 38 lotus (Nelumbo nucifera Gaertner) cultivars were evaluated and quantified by ultra-performance liquid chromatography with photodiode array (UPLC-PDA) and ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS) for the first time. In total, twenty flavonoids were detected and identified, among which fourteen were found to be flavonoid C-glycosides. These cultivars were clustered into three clusters characterized by sum of detected flavonoids. Furthermore, our study showed that the correlation between total polyphenol content and antioxidant activity which was measured by DPPH and FRAP assays was significant positive. Among them, ‘Taikonglian’, ‘Jinqi’, ‘Yinqiu’, and ‘Hongtailian’ exhibited the greater potential of antioxidant activity and could be utilized in the production of healthcare products. This research is of great value to more comprehensive understanding of medicinal property of lotus plumule. Also, it will be helpful in the development of commercial exploitation of lotus plumule.