Objective To establish and optimize ISSR-PCR systems of Chrysanthemum morifolium and lay the foundation for its genetic diversity research. Methods Based on the analysis of variance, an orthogonal design was used to optimize the ISSR-PCR amplification system on C. morifolium by four factors (Taq polymerase, Mg2+, dNTP, and primer) at three concentration levels, respectively. Results A suitable ISSR reaction system was constructed with the 20 ?L reaction system containing 1.00 U Taq polymerase, 2.00 mmol/L Mg2+, 0.20 mmol/L dNTP, and 0.50 ?mol/L primer. Conclusion ISSR-PCR is significantly influenced by the concentration of Taq polymerase, Mg2+, and dNTP. This ISSR-PCR system could provide clear bands, reliable reaction system, and abundant polymorphisms . It is proved to be suitable for the study of the genetic diversity of C. morifolium