The orthogonal design was used to optimize ISSR-PCR amplification system of Pyrus bretschneideri Rehd.cv.Yali Hc in four factors(DNA template,primer,dNTPs,and Taq DNA polymerase)at five levels respectively.The results of PCR was analyzed by software DPS 7.55,and the best level of each factor were selected.The most suitable ISSR-PCR reaction system was 60 ng DNA template,0.4μmol/L primer,0.1mmol/L dNTPs,2.0U Taq DNA polymerase in 20 μL reaction system.