Abstract: The gypsy moth, Lymantria dispar Linnaeus (Lepidoptera: Lymantriidae), is a destructive defoliator with a broad host range. Studying genetic variation is a helpful tool for revealing the genetic differentiation of different geographic populations of gypsy moth. 5 main factors of ISSR reaction system for gypsy moth (Lymantria dispar Linnaeus) was optimized with sample from Hebei Province, China. Factors with 8 concentration gradients were performed successively. Each optimum concentration of a factor was applied to the next optimization experiment. The final ISSR reaction system for gypsy moth was as follows: 0.5 U Taq DNA polymerase, template DNA 1.5 ng/μL, primer 2.4μmol/L, 1.75 mmol/L MgCl2, 0.28 mmol/L dNTPs. Then ddH2O was added to the final volume of 20μL. This optimum reaction system would lay fundamental basis for the genetic diversity and variation research for gypsy moth.