AbstractXibei tree peony is a distinctive cultivar group that features red–purple blotches in petals. Interestingly, the pigmentations of blotches and non-blotches are largely independent of one another. The underlying molecular mechanism had attracted lots of attention from investigators, but was still uncertain. Our present work demonstrates the factors that are closely related to blotch formation inPaeonia rockii‘Shu Sheng Peng Mo’. Non-blotch pigmentation is prevented by the silencing of anthocyanin structural genes, among whichPrF3H,PrDFR, andPrANSare the three major genes. We characterized two R2R3-MYBs as the key transcription factors that control the early and late anthocyanin biosynthetic pathways. PrMYBa1, which belongs to MYB subgroup 7 (SG7) was found to activate the early biosynthetic gene (EBG)PrF3Hby interacting with SG5 member PrMYBa2 to form an ‘MM’ complex. The SG6 member PrMYBa3 interacts with two SG5 (IIIf) bHLHs to synergistically activate the late biosynthetic genes (LBGs)PrDFRandPrANS, which is essential for anthocyanin accumulation in petal blotches. The comparison of methylation levels of thePrANSandPrF3Hpromoters between blotch and non-blotch indicated a correlation between hypermethylation and gene silencing. The methylation dynamics ofPrANSpromoter during flower development revealed a potential early demethylating reaction, which may have contributed to the particular expression ofPrANSsolely in the blotch area. We suggest that the formation of petal blotch may be highly associated with the cooperation of transcriptional activation and DNA methylation of structural gene promoters.