DNAs were extracted from 99 endophytic fungi isolated from Huperzia crispata and the best annealing temperatures of 10 primers (UBC842, UBC848, UBC850, UBC856, UBC861, UBC864, UBC868, UBC873, UBC880 and UBC887) were determined for optimization of ISSR-PCR amplification of endophytic fungi from Huperzia crispate through single factor test and orthogonal design. The result showed that the optimal ISSR–PCR reaction volume was 25 μL, containing 2.5 μL of 10×PCR Buffer (2.0 mmol/L Mg2+), 0.6 μL of dNTPs (10 mmol/L), 2.0 μL of primer (10 pmol/μL), 1.5 μL of Taq E (1U/μL), 3 μL of template DNA (10 ng/μL) and 15.4 μL of aseptic ddH2O. PCR using primer UBC873 conducted on 99 endophytic fungi showed that the genetic polymorphism for endophytic fungi of Huperzia crispata was abundant.