Aquilaria sinensis(Lour.)Gilg classified as Thymelaeaceae, is one of the valuble medicinal plants in our country. But its wilding resource has been profoundly damaged, and has been recorded in《China Plants Red Book》. The improved CTAB method which can extracting high-quality genome DNA of Aquilaria sinensis(Lour.) Spreng was much more simple and efficient than the conventional methods. And the extracted DNA was suitable for the polymorphic analysis based on PCR, such as ISSR-PCR. The optimal ISSR-PCR reaction conditions in Aquilaria sinensis(Lour.) Spreng was established by studying the main parameters. Results showed that the optimum concentration of five important components i.e. template DNA, primer, Mg2+, dNTP, Taq DNA polymerase in 20 µl reaction mixture was 25ng、0.8µmol/L、2.5mmol/L、0.2mmo1/L、1.0U, respectively, and at least 35 PCR cycles should be carried out to ensure sufficient PCR products.