Abstract: The genome DNAs of 16 strains of Rhizoctonia solani isolated from tobacco target spot were extracted by the improved CTAB method. The OD260/OD280 values of the obtained DNAs ranged from 1.76 to 1.89, the electrophoretic bands were clear and free of dispersion and tailing, the DNAs were of better quality. The main feators influencing RAPD-PCR reaction, including the concentrations of Mg²⁺, dNTP and primer, and the dosages of template DNA and Taq DNA polymerase, were optimized by the combination of single factor and orthogonal experiments and an optimal RAPD reaction system was established. The total volume of the optimal reaction system was 25 μL, including 10 ng template DNA, 3.25 mmol/L Mg²⁺, 0.175 mmol/L dNTP, 1.4 U Taq DNA polymerase and 0.5 μmol/L primer. Clear, stable and specific bands and satisfactory amplification results were obtainable by the optimized reaction system.