Abstract: The factors influencing ISSR—PCR experiments to analyze the genetic divergence in: cycas parz hihuaensis were investigated. Comparative experiments on the concentrations of template DNA, ISSR primer, Taq DNA polymerase and the annealing temperature were carried out. The results showed that the optimum reaction condition of ISSR experiment was 15~25ng DNA template, 2.0~2.5mmol/Lmg2+, 0.2mmol/LdNTPs, 0.2μmol/L ISSR primer, 0.2U Tag DNA polymerase, annealing temperature of 48~55℃ and 35 cycles in each reaction system of 20μl, which set up a basis for the study of genetic divergence among Cycas panzhihuaensis.