Abstract: Abstract: To optimize the inter simple sequence repeat（ISSR）reaction condition for Erythropsis kwangsiensis genomic DNA, the concentrations of MgCl₂, dNTPs, Taq polymerase and template DNA were studied with an orthogonal experimental design, and the optimal anneal temperature of primer and cycles were determined through gradient PCR. The optimal PCR system for ISSR analysis was 1×PCR buffer, 2.5 mmol/L MgCl₂, 0.15 mmol/L dNTPs, 0.06 U/μL Taq polymerase, 3 ng/μL template DNA, 0.2 μmol/L primer in 25 μL reaction solution. The augmentation procedure was pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 45 s, annealing at 52℃ for 45 s, extension at 72℃ for 1.5 min, reaction with 30 cycles, and extension at 72℃ for 7 min and holding the samples at 4 ℃. The system was applied in the amplification of 10 varieties of Erythropsis kwangsiensis, and indicated the suitability and stability of the system.