Herbaceous peony (Paeonia lactiflora Pall.) is an important ornamental plant which contains different flower colors. In this paper, eight genes encoding phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose: flavonoid 3-o-glucosyltransferase (UF3GT) were isolated. Moreover, the expression patterns of these eight genes and UF5GT in the flowers were investigated in three cultivars, that is, 'Hongyanzhenghui', 'Yulouhongxing' and 'Huangjinlun' with purplish-red, white and yellow flower respectively. Furthermore, flavonoid accumulation in the flowers was also analyzed. The results showed that in different organs, most of genes expressed higher in flowers than in other organs. During the development of flowers, all genes could be divided into four groups. The first group (PlPAL) was highly expressed in S1 and S4. The second group (PlCHS and PlCHI) was at a high expression level throughout the whole developmental stages. The third group (PlF3H, PlF3'H, PlDFR, PlANS and PlUF5GT) gradually decreased with the development of flowers. The fourth group (PlUF3GT) gradually increased during the flower development. In addition, anthoxanthins and anthocyanins were detected in 'Hongyanzhenghui' and 'Yulouhongxing', chalcones and anthoxanthins were found in 'Huangjinlun'. When different color flowers were concerned, low expression level of PlCHI induced most of the substrate accumulation in the form of chalcones and displaying yellow, changing a small part of substrates to anthoxanthins, and there was no anthocyanin synthesis in 'Huangjinlun' because of low expression level of DFR. In 'Yulouhongxing', massive expressions of upstream genes and low expression of DFR caused synthesis of a great deal of anthoxanthins and a small amount of colorless anthocyanins. In 'Hongyanzhenghui', a large number of colored anthocyanins were changed from anthoxanthins because of PlDFR, PlANS and PlUF3GT high expressions. These results would provide us a theoretical basis to understand the formation of P. lactiflora flower colors.