Abstract: In order to establish a reproducible, stable, and suitable reaction system of Saccharina for ISSR analysis of genetic differences, the genomic DNA extracted from gametophytes of Saccharina was used as template, the different concentrations of the factors in reaction system were optimized by orthogonal design experiment. The results showed that, the optimal ISSR reaction system (20 μL) contained 1 ×PCR buffer, 1.2 mmol/L Mg2 , 0.2 mmol/L dNTPs, 1 μmol/L ISSR primer, 40 ng template DNA and 0.25 U Taq DNA polymerase. The PCR program was pre-denatured at 95℃ for 3 min and followed by 38 cycles of 45 s at 95℃, 45 s at 52℃, 2 min at 72℃, and final extension of 10 min at 72℃. The optimal ISSR reaction system was stable and repeatable. This system could provide the technical foundation for the application of ISSR molecular marker technology for the study on genetic diversity and molecular identification ofSaccharina .