Abstract: Based on the genomic DNA extracted from honeysuckle, the factors influenced ISSR were optimized and the effect of 6 factors such as annealing temperature, Taq DNA polymerase dosage，DNA templates concentration，primer concentration, dNTPs concentration and Mg2+ concentration on ISSR amplification were tested using single factor experiment. A reaction system and amplified procedure suitable for honeysuckle were established, that is 20 μL amplification reactions system containing 1×PCR buffer（Mg2＋free）, 1.5 U Taq DNA polymerase, 0.15mmol·L-1 dNTPs, 0.4 μmol·L-1 primer, 1.5 mmol·L-1 MgCl2, 60 ng template DNA. The optimized annealing temperature is 49.9℃. The optimal amplified procedure was as follows: after a pre-denaturing of 5 min at 94℃, 35 cycles were performed with 30 s for denaturing at 94℃ , annealing of 30 s at 49.9℃, extension of 1.5 min at 72℃ , 7 minutes of extension at 72℃ in the final cycle and hold at 4℃. Using these optimal amplification conditions, 10 stable and repeatable ISSR primers were selected from total 100 primers. The genome DNA of 22 honeysuckle cultivars were amplified with 10 ISSR primers and 108 bands were amplified totally, including 96 polymorphism bands with polymorphism rate of 88.9%. The establishment of the ISSR-PCR reaction system could settle favorable foundation for identification of honeysuckle cultivars ,classification and analysis of the genetic diversity of honeysuckle using ISSR molecular marker techniques．