Abstract: The present study was aimed to establish and optimize SCoT-PCR amplification system of litchi, and conduct the identification and genetic diversity analysis of‘Ziniangxi’,‘Wuheli’, and their hybrids. Orthogonal design was applied to optimize litchi SCoT- PCR amplification system in five factors, including rTaq DNA polymerase, Mg2 + , DNA template, dNTPs and primers. The optimum reaction system (20μL) contained 30 ng template DNA, 1 U rTaq polymerase, 0.75μmol/L primers, 3 mmol/L Mg2+ and 0.3 mmmol/L dNTPs. The most suitable annealing temperature of primers was 50.6℃. Genetic analysis of‘Ziniangxi’,‘Wuheli’, and their hybrids were carried out using this SCoT-PCR system. A total of 280 loci were obtained using 32 SCoT primers and 131 loci were polymorphic. The real hybrids could be identified by SCoT. 60 hybrids can be identified by four SCoT primers. UPGMA cluster analysis and genetic similarity coefficient showed that the similarity coefficient of 30 hybrids ranged from 0.406 to 0.867, and 30 progenies could be grouped into three distinct families based on similarity of 0.68, indicating that it possessed rich genetic diversity. The optimum SCoT-PCR system of litchi was stable and very useful for genetic diversity analysis of litchi, which would possess considerable potential for litchi breeding.