Abstract: In order to establish a suitable SRAP reaction system, the orthogonal experiment was used to establish and optimize the five main factors (dNTPs, Mg²⁺, Taq DNA polymerase, primer, and DNA template) of sequence-related amplified polymorphism (SRAP) system with young leaves of tobacco mutant SN01 resistant to Potato virus Y-vein necrosis strain (PVY^N). The results showed that the best amounts of factors in 20 μL of the reaction system were: dNTPs (2.5 mmol/L) 3.2 μL, Mg²⁺ (25 mmol/L) 1.6 μL, Taq DNA polymerase (2.5 U/μL) 0.16 μL, primer (10μmol/L) 0.6 μL, DNA template 120 ng, respectively. The best annealing temperature of 57 ℃ and cycle times for 35 of the SRAP program were also conducted. This optimized system provides reliable basis for further study on the resistance genes of tobacco resistance mutant SN01.