Abstract: The aim was to establish a stable, reproducible, and suitable reaction system of Lilium for ISSR analysis of genetic differences. PCR amplifications were carried out until the different concentrations of the factors in the ISSR reaction system were designed, and based on the principle of high stability and reproducibility, the stable and reliable ISSR reaction system was eventually established after a series of adjustment and optimization of the reaction system. The optimum ISSR-PCR reaction system (25μL) contained 40 ng DNA template, 2.0 mmol/L Mg²⁺, 1.5 U TaqDNA polymerase, 0.8μmol/L ISSR primer and 200μmol/L dNTPs. The reaction mixtures were pre-denatured at 94℃for 5 min and subjected to 35 cycles of 1 min at 94℃, 1 min at 51.8℃, 2 min at 72℃, and a final extension step of 8 min at 72℃. The ISSR-PCR systems showed stable reaction system, better repeatability, and reliable abundant polymorphisms. This reaction system could provide a technological base for the study on genetic polymorphism analysis, cultivar identification of Lilium at the molecular level.