Abstract: ‘Constanta’ and ‘Sorbonne’ and 9 F1 hybrids of ‘Constanta’ × ‘Sorbonne’ were used as materials in this experiment, and a modified method of CTAB was applied to extract the genomic DNA from the young leaves. The orthogonal design L16（45）was used to optimize the ISSR-PCR system for oriental lily at four levels and five factors, namely, DNA polymerase, Mg2+, DNA template, dNTPs and primers. The results of PCR were evaluated by intuitive and variance analysis. Results showed that the best reaction system was 20 μL reaction solution, containing 0.5 μmol/min Taq DNA polymerase, 1.75 mmol/mL Mg2+ , 40-100 ng template DNA, 0.15 mmol/mL dNTPs, 0.6 μmol/L primers, 2 μL 10×PCR buffer and ddH2O. Using single factor experiment design, we found that 55-45 ℃ was the best touch down PCR annealing temperature, the best cycle times was 30 and the best extend time was 15 min. The stabilization of the optimized reaction system was tested by using ‘Constanta’ and ‘Sorbonne’ and 9 F1 hybrids of ‘Constanta’ × ‘Sorbonne’.