Wild rice Zizania latifolia Griseb (Turcz), an aquatic/wetland genus, is a member of tribe Oryzeae widely distributed across China. Z. latifolia is not only an important genetic resource for crop improvement but also contributes significantly to the ecosystem. However, studies on Z. latifolia have been slow because of the lack of methodology and knowledge in all aspects. In this work, we first tried to develop and establish an optimized ISSR-PCR reaction system for genotyping of Z. latifolia using the single factor variable method in which 6 factors (dNTPs, Mg2+, Taq polymerase, primer, template DNA and primer annealing temperature) at 12 different concentrations or gradients were tested. We finally set up an optimized 20 μL reaction system as follows: 0.5 mM dNTPs, 3.0 mM Mg2+ (10× Buffer), 0.24 U·μL-1Taq polymerase, 1.0 μM primer，1.5 ng·μL-1DNA template and annealing temperature at 55℃. The optimized ISSR system was successfully applied in the amplification of 80 Z. latifolia individual samples, thus, the adaptability and stability of the system were testified.