Abstract: The orthogonal design and single factor test were used to optimize and study the suitable system in TRAP reaction of Lactuca sativa L. There were four factors which influenced the effects of TRAP- PCR including Mg²⁺, Taq DNA polymerase, dNTPs and primers. The optimal reaction system for TRAP reactions in leaf lettuce was established, which was in 20μL reaction volume containing 60-100 ng DNA, 2μL 10×PCR Buffer, 2.0 mmo/L Mg²⁺, 1 U Taq DNA polymerase, 0.2 mmol/L dNTPs and 0.75μmol/L primers. The result of amplified production was stable, high definition and polymorphic, indicating that the reaction system was the best to analyze genetic diversityandrelationship in leaf lettuce by TRAP .