Herbaceous peony(Paeonia lactiflora Pall.) is a famous flower with medicinal values, and its flowers have a number of medicinal constituents, especially flavonoids. In this study, a P. lactiflora cultivar with double colors including white outer-petal and yellow inner-petal was used as the experimental materials to perform the qualitative and quantitative analysis of flavonoids by high-performance liquid chromatograph-electrospray ionization-mass spectrometry(HPLC-ESI-MSn) and investigate the expression patterns of flavonoid biosynthetic genes using real-time quantitative polymerase chain reaction(Q-PCR). The results showed that the colors of both petals gradually weakened with flower development. Moreover, one main anthocyanin composition(peonidin 3,5-di-O-glucoside) and five main anthoxanthin compositions(kaempferol di-hexoside, kaempferol-3-O-malonylglucoside-7-O-glucoside, quercetin-3-O-galactoside, luteolin-7-O-glucoside and isorhamnetin-3-O-glucoside) were found in the both, differing significantly in their peak areas only. Total anthocyanin, anthoxanthin and flavonoid contents in white outer-petal and yellow inner-petal gradually decreased during flower development, and were consistently higher in white outer-petal. Furthermore, the expression patterns of nine structural genes in P. lactiflora flavonoid biosynthetic pathway showed that the expression levels of phenylalanine ammonialyase gene(Pl PAL), chalcone synthase gene(PlC HS), flavanone 3-hydroxylase gene(PlF 3H), anthocyanidin synthase gene(PlA NS) and UDP-glucoside: flavonoid 5-O-glucosyltransferase gene(Pl F5GT) in two petals basically presented declined tendencies, and transcription levels of Pl PAL, Pl CHS, Pl ANS, Pl F3 GT and Pl F5 GT also tended to be higher in white outer-petal, which was correlated with their flavonoid contents. These results would lay a solid foundation for the exploration and utilization of flavonoid resources in P. lactiflora flowers. Herbaceous peony (Paeonia lactiflora Pall.) Is a famous flower with medicinal values, and its flowers have a number of medicinal constituents, especially flavonoids. In this study, a P. lactiflora cultivar with double colors including white outer-petal and yellow inner- petal was used as the experimental materials to perform the qualitative and quantitative analysis of flavonoids by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MSn) and investigate the expression patterns of flavonoid biosynthetic genes using real-time quantitative polymerase chain reaction (Q-PCR). The results showed that the colors of both petals gradually weakened with flower development. Moreover, one main anthocyanin composition (peonidin 3,5-di- O-glucoside) and five main anthoxanthin compositions (kaempferol di- hexoside, kaempferol-3-O-malonylglucoside-7-O-glucoside, quercetin-3-O-galactoside, luteolin- 7-O-glucoside and isorhamnetin-3-O-glucoside were found in the both, diff ering significantly in their peak areas only. Total anthocyanin, anthoxanthin and flavonoid contents in white outer-petal and yellow inner-petal gradually decreased during flower development, and were consistently higher than white outer-petal. Further, the expression patterns of nine structural genes in P. lactiflora flavonoid biosynthetic pathway showed that the expression levels of phenylalanine ammonialyase gene (Pl PAL), chalcone synthase gene (PlC HS), flavanone 3-hydroxylase gene (PlF 3H), anthocyanidin synthase gene (PlA NS) and UDP-glucoside : flavonoid 5-O-glucosyltransferase gene (Pl F5GT) in two petals substantially presenting tendencies, and transcription levels of Pl PAL, Pl CHS, Pl ANS, Pl F3 GT and Pl F5 GT also tended to be higher in white outer-petal , which was correlated with their flavonoid contents. These results would lay a solid foundation for the exploration and utilization of flavonoid resources in P. lactiflora flowers.