Abstract: The coconut ISSR-PCR reaction system was optimized, we distilled the total DNA of two coconut cultivars that were Hainan Tall and Malaysia Tall, and the single factor experiment was designed which included five factors with five different concentrations and then the effect of the variations of these concentrations were studied. Meanwhile, we obtained the annealing temperature of primer 807, the result shows the optimal annealing temperature was 56℃, at last, the optimum reaction system for coconut consists of 0.5~2.0U Taq DNA polymerse, 2.5~3.0mmol/L 1 0×Taq Buffer(25mmol/L Mg2＋), 50ng template DNA, 0.2~0.5mmol/L dNTPs and 0.8~1.6μmol/L primer with total 20μL reaction solution.