To seek a standardizing program of ISSR technique for genetic diversity analysis of Thespesia populnea,a single factor experiment was designed to optimize ISSR-PCR amplification system.The suitable reaction system was obtained,that is 20 μL reaction system containing 2.0 μL of 10×Buffer,27.5 ng of total DNA,2.0 μL of dNTP ,1 U Pyrobest DNA polymerase and 1.25 μmol/L of ISSR primer.The profile of ISSR-PCR was an initial denaturation step for 5 min at 94℃,follow by 35 cycles of 1 min at 94℃,45 s at annealing temperature 49℃,1 min at 72℃,and a final elongation 10 min at 72℃ for one cycle,then termination reaction at 4℃.Ten polymorphic primers were screened using this reaction system.Stable and clear amplification patterns were obtained,indicating the ISSR-PCR amplification system was feasible.The factors affecting the amplification of genome DNA of Thespesia populnea were also discussed in the paper.It provides the basis on studies of germplasm resources and genetic diversity of T.populnea.