Based on screening several key reaction conditions of ISSR (inter-simple sequence repeat) markers,such as the concentrations of template DNA,primers and Mg2+,an optimal ISSR reaction system for Chimonanthus nitenswas established,namely,40 ng genomic DNA,0.3 μmol/L primer,1.5 mmol/L Mg2+,1 U Taq,0.2 mmol/L dNTP,and 2.5 μL 10×buffer were contained in 25 μL reaction system.Using this optimal ISSR reaction system,12 polymorphic primers were screened out.Furthermore,the best annealing temperature of the every selected primer was decided according to the amplified results with different temperatures among 51 ℃ and 56 ℃.