The effects of various factors on ISSR-PCR reaction, such as concentration of Mg2+, dNTPs and primers, DNA template and Taq DNA polymerase, were investigated to optimize the ISSR-PCR reaction system of Freesia refracta through orthogonal tests. The optimized ISSR-PCR system of F. refracta incloded 1×Taq DNA polymerase buffer (10 mmol·L-1 KCl, 8 mmol·L-1(NH4)2SO4, 10 mmol·L-1 Tris·HCl, pH 9.0, 0.05% NP-40), 2 mmol·L-1 MgCl2, 0.06 U·μL-1 Taq DNA polymerase, 0.4 μmol·L-1 primer, 4.0 ng·μL-1 DNA template, 0.6 mmol·L-1 dNTPs in 25 μL PCR reaction system. By temperature gradient PCR, the optimum annealing temperature was determined as 51.5℃. The optimized system laid the groundwork for ISSR molecular analysis of F. refracta.