Abstract: The study focuses on the influential factors of ISSR experiments used to analyze Korean pine. Comparative experiments on DNA extraction and PCR amplification among fresh leaves, dry leaves, embryos and endosperms were conducted. The results showed that the four kinds of materials were all feasible. The effect of purity and concentration of template, dNTP and TaqDNA polymerase on amplification was analyzed. Different annealing temperature, extension time and cycle times were also compared. 17 primers with stable amplification and rich polymorphism for ISSR were screened. The reaction condition of ISSR experiment of Korean pine was discussed, which was a basis for the study on genetic divergency among Korean pine populations.