Abstract: To explore the mechanism hydroxysafflor yellow A (HSYA) biosynthesis and regulation, the effect of methyl jasmonate (MeJA) treatment on gene expression related to the biosynthesis of hydroxysafflor yellow A (HSYA) was analyzed, and expression differences in genes involved in HSYA biosynthesis in safflower of different colors was quantified. MeJA at concentrations of 0, 50, 100, and 200 μmol·L^-1 was sprayed onto safflower florets to determine the optimal concentration of MeJA. Safflower was treated with 100 μmol·L^-1 MeJA and florets were harvested 0, 3, 6, 12 and 24 h after treatment. The content of MeJA was determined by high performance liquid chromatography (HPLC). RNA was extracted from safflower florets treated with 100 μmol·L^-1 MeJA for 6 h. The transcription of key genes involved in the biosynthesis of HSYA was quantified by qRT-PCR and differentially expressed genes were identified. The content of HSYA increased after treatment with MeJA, with 100 μmol·L^-1 MeJA treatment for 6 h having the greatest effect on HSYA accumulation. qRT-PCR results showed that MeJA could significantly increase the transcription of HSYA biosynthesis genes including PAL2, PAL4, 4CL2, 4CL4, 4CL5, CHS3, CHS4 and CHI2. The content of HSYA differed between safflowers of different colors with a trend of red > orange-yellow > yellow > white. The results of qRT-PCR showed that the expression of CHS1 and CHI2 in red, orange and yellow safflower was significantly higher than that in white safflower. These results indicate that MeJA promotes the accumulation of HSYA by up-regulating the expression of genes involved in the biosynthesis of HSYA such as PAL2, PAL4, 4CL2, 4CL4, 4CL5, CHS3, CHS4 and CHI2, and the variation of HSYA content in safflower of different colors was related to a difference in the level of expression of CHS1 and CHI2.