Abstract: ObjectiveThis research was conducted to establish a ISSR-PCR reaction system for the paternity indenti-fication and genetic diversity analysis of Tripterygium wilfordii Hook. f.MethodSingle-factor analysis method was applied to optimize and select the main components and annealing temperature of five PCR reaction system having effects on PCR amplification of the 32 T. wilfordii samples,including Mg2+,dNTPs,Taq DNA polymerase,primer concentration and template DNA content. And the 32 samples were used to testify the optimized ISSR-PCR reaction system.ResultAn ISSR-PCR reaction system applicable for indentifying germplasm resources of T. wilfordii was established:20.00μL reac-tion mixture contained 2.00 mmol/L Mg2+,0.30 mmol/L dNTPs,1.50 U Taq DNA polymerase,0.40 mol/L primer,20.00 ng template DNA and 2.00μL 10 × Buffer. The optimal annealing temperature was 54.7℃.ConclusionThe T. wilfordii ISSR-PCR reaction system established is of high stability,repeatability and applicability,indicating this reaction system is suitable for the genetic diversity analysis of T. wilfordii from different geographical provenances.