Abstract: This study established and optimized SSR-PCR reaction system of Fraxinus, and used this system selected 3 primer pairs from 50 primer pairs for the SSR analysis based on their reproducible and clear banding patterns. These primer pairs could be used for further research. This experiment established through L₁₆(4⁵) orthogonal design which selected from 4 levels of 5 factors (Taq DNA polymerase, DNA, dNTPs concentration, primers concentration and Mg²⁺ concentration) in SSR- PCR system and optimized through single factor experiment about the main factors of the PCR reactions. The optimal SSR-PCR reaction system consisted of Mg²⁺ (25 mmol/L) 0.8μL, containing forward- and reversed- primer (10μmol/L) 0.2μL respectively, dNTP (10 mmol/L) 0.3μL, Taq DNA polymerase (5 U/μL) 0.05μL、DNA (5-10 ng/μL) 2.00μL, 10×PCR Buffer 1.0μL and ddH₂O 5.45μL. The reaction system was the most conformable one for Fraxinus’SSR-PCR, and was established as the good foundation of SSR-PCR marker technique on Fraxinus.