Abstract: Factors affecting the Inter-Simple Sequence Repeats (ISSR) analysis of longan,such as annealing temperature and concentrations of template DNA,Mg2 + and dNTP were examined.The optimal ISSR-PCR reaction system (25 μL) was found to be: 20 ng template DNA,1.5 U Taq DNA polymerase,0.7 μmol·L-1 primer,2.0 mmol·L-1 Mg2+,and 0.2 mmol·L-1 dNTPs.Using the single primer (ISSR-13),fingerprints of 12 genotypes were constructed from different origins with their abundant polymorphism of DNA spectra.The results suggested that ISSR-PCR could be a suitable reaction system for studying longan genetic diversity.