Abstract: In this study, one-factor experimental design had been used to establish the SRAP-PCR reaction system of sunflower, which including several different gradients of Mg2+, dNTPs, primer concentration, Taq DNA Polymerase, template DNA and filter out the suitable range, on this basis, then optimize the five factors which influence the SRAP-PCR through the orthogonal experimental design of L16(45). Eventually brought about an optimized system for sunflower, which was as follows: 20 μL capacity of reaction system contains 10×Buffer 2 μL, Mg2+ 2.75 mmol/L, dNTPs 0.18 mmol/L, Taq DNA Polymerase 1.25 U, forward primer and reverse primer 0.3 μmol/L, template DNA 60 ng, the best annealing temperature 52.2℃. Finally, 22 varieties of sunflower were used to test the optimized PCR reaction system stability. The results showed that, the bands of the amplification clear and high polymorphism, so the system was reliable which could be applied in the germplasm identification and the genetic map construction of sunflower effectively.