Abstract: An orthogonal design was used to optimize an ISSR(inter-simple sequence repeats)-PCR(polymerase chain reaction) amplification system for Oxalis triangularis, involving five levels of five main factors (Mg²⁺, Taq DNA polymerase, primer, template DNA and dNTP). The annealing temperature of suitable primers, which were selected from 100 random ISSR primers, was optimized and the reaction program was improved so that a suitable ISSR-PCR reaction system for O. triangularis was established. The reactions were performed in a 25 μL volume containing 2.5 μL 10×buffer, 1.25 mmol/L Mg²⁺, 0.9 U Taq DNA polymerase, 0.3 μmol/L primer, 60～100 ng template DNA, and 0.25 mmol/L dNTP. The optimized reaction program was: 94℃ for 5 min, followed by 40 cycles of 94℃ for 50 s, annealing at a suitable temperature for different primers for 60 s, 72℃ for 1.5 min, extension at 72℃ for 7 min and preservation at 4℃. The establishment of this system provides a theoretical basis and technical references for further research on the genus Oxalis by ISSR markers.