The orthogonal design involving four levels of four main factors (dNTPs, primer, Taq DNA polymerase, and DNA template) was used to optimize ISSR–PCR system on Heterodera elachista. The results showed that the optimal 25 μL ISSR–PCR system contained 0.3 μL Taq DNA polymerase (5 U/μL),1 μL template DNA, 2 μL dNTPs (2.5 mol/L), 1 μL primer (10 μmol/L), 2.5 μL 10×PCR Buffer（20 mmol/L Mg2+ plus）2.5 μL and 18.2 μL ddH2O. And the optimal annealing temperature of primer UBC887 was 48 ℃. The optimized ISSR–PCR for H. elachista has good repeatability and stability.