Abstract: The study aims to optimize ISSR-PCR reaction system in Pyrus and verify its universality in other markers. Taking‘Shinseiki’בChonghuadali’F1 hybrid generation as the experimental material, the L₂₅(5⁶) orthogonal design was used to optimize 4 factors in ISSR-PCR amplification, including DNA template, primer, dNTPs and Taq polymerase. The result of PCR was analyzed, the most suitable ISSR-PCR system for Pyrus was established, which was 20μL reaction system, containing DNA 3 ng/μL, dNTPs 0.20 mmol/L, primer 0.5μmol/L and TaqDNA polymerase 0.03 U/μL. Optimization of the reaction system also applied to SRAP and SSR markers. The study can provide theoretical basis for future genetic diversity analysis, evaluation of germplasm resources and genetic map construction in Pyrus, and cut the cost and raise the efficiency of the experiment.