Abstract: The aim of this study was to optimize the RAPD reaction system in poplar. The L₁₆(⁴⁵) orthogonal tests design and two single factors tests were used to study the appropriate concentrations of the main factors with Populus alba. The results showed that an optimized RAPD reaction system was established: the total 20μL contained Mg²⁺ 1.5 mmol/L, Taq polymerase 2.0 U, primer 0.2μmol/L, dNTPs 0.2 mmol/L, DNA template 50 ng. Amplified procedure was: 94℃for 3 min, 38 cycles at 94℃for 40 s, 38℃for 60 s, 72℃for 90 s, followed by 72℃for 7 min and then ramped to 4℃to hold the reaction. Amplified products were detected by electrophoresis in 1.5% agarose gel. It was evident that the reaction system established in this study was stable and reliable for RAPD analysis, and could be used on genetic analysis of poplar.