Abstract: In order to establish and optimize the SSR-PCR reaction system of Orychophragmus violaceus, the L25 (56) orthogonal design was applied to optimize the five main factors (DNA template, Mg2 , dNTPs, primers and Taq enzyme) at five levels of SSR-PCR reaction system in this study. The PCR result was analyzed to select the best levels of the five factors, and a suitable SSR reaction system for Orychophragmus violaceus was established. The result showed that the optimal SSR-PCR system of 10 μL volumes consisted of 2.0 mmol/L Mg2 , 0.4 mmol/L dNTPs, 0.6 μmol/L primer, 20 ng DNA template, and 0.6 U Taq polymerase. The optimized SSR-PCR reaction system was used to verify with three Orychophragmus violaceus germplasms, 12 primer pairs were selected with clear banding patterns and abundant polymorphism bands from 50 primer pairs, and the result showed that the optimal reaction system had excellent stability and generality. The establishment of SSR-PCR reaction system provides a theoretical basis and technical references for molecular biology research on Orychophragmus violaceus.