Abstract: The objective of this study was to establish the optimal SRAP-PCR (sequence-related amplified polymorphism polymerase chain reaction) reaction system of Bletilla striata, in order to provide the technical support for the molecular genetic evaluation of Bletilla striata in different geographic populations at the molecular level. The tender leaves of Bletilla striata collected from Anlong County of Guizhou Province were used as the materials. The orthogonal design (L₉(3⁴)) was applied to optimize the dosage of four factors (primer, Taq DNA polymerase, Mg²⁺ and dNTPs) in the reaction system. The primer combination for the orthogonal design was (Me1 / Em1). Then, the optimal SRAP-PCR reaction system for Bletilla striata was established and the primers were screened, thus to screen the primer combination with high stability and good polymorphism. The results showed that the optimal SRAP-PCR reaction system (25 μL) for Bletilla striata was as follows: 2.50 μL 10×Buffer, 2.50 mmol·L⁻¹ Mg²⁺, 0.30 mmol·L⁻¹ dNTPs, 0.25 μmol·L⁻¹ primers, 1.50 U Taq DNA polymerase and 20-80 ng template DNA. The stability of the optimal SRAP-PCR reaction system was detected by using 21 Bletilla striata, and the system had high stability and good repeatability. By taking the genomic DNA of Bletilla striata as the template, 80 pairs of primer combinations were screened by using the best SRAP-PCR reaction system. Finally, 13 pairs of polymorphic primers were obtained, and 182 loci were amplified, of which 152 were polymorphic loci, and the percentage of polymorphic loci was 83.52%. The system had high stability and good repeatability, which provided relevant technical support for the subsequent genetic diversity research.