Wheat (Triticum aestivum Linn.; Poaceae), one of the most popular food crops worldwide, contains basic and essential nutrients and various health benefiting phytochemicals. Among them, flavonoids have attracted significant interest owing to their various health-promoting properties. In this study, 35 wheat mutant lines were developed via gamma-irradiated mutation breeding from the original cultivar. The effects of radiation breeding on the endogenous phytochemical production in the sprouts of these mutant lines were investigated using high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS) with multivariate analysis for the first time in this study. Fourteen characteristic peaks, including eleven flavone C-glycosides, two flavone O-glycosides, and one flavone, were identified. In addition, the contents of three flavone C-glycosides, namely, isoschaftoside, isoorientin, and isoscoparin, in 37 wheat sprout samples from the original cultivar, certificated cultivar, and the mutant lines were determined. A heat map combined with hierarchical clustering showed variation in the relative content for the flavonoids between the 37 wheat sprout samples, clustering into three groups. On principal component analysis scores scatter and loading plots, significant differences in the levels of flavonoids were found between the samples and several markers responsible for group separation were detected. These results provide a scientific reference for the phytochemical variation in wheat mutant lines, thereby aiding in further mutation mechanism studies and for the quality control of the improved wheat cultivars.