The stem of caulis spatholobi was used as the experimental material for DNA extraction using the improved CTAB method.The ISSR-PCR reaction system was optimized and constructed using orthogonal and single factor experiments.The results showed that the optimal ISSR-PCR reaction system for caulis spatholobi was DNA template 60 ng,Taq enzyme 2.00 U,Mg2+ concentration 1.75 mmol/L,dNTP concentration 0.25 mmol/L,primer concentration 0.20 μmol/L,and optimal annealing temperature 44.6℃.20 ISSR primers suitable for caulis spatholobi from were selected 100 ISSR primers.