In this study, inter-simple sequence repeat (ISSR) was evaluated for its potential use in the identification of 54 Osmanthus fragrans cultivars. 13 out of 70 (18. 6%) ISSR primers could generate reproducible polymorphic fragments. The ISSR-PCR assay revealed a total number of 90 DNA bands, of which 79 bands were polymorphic (the percentage of polymorphic bands, PPB=89. 9%). Optimized ISSR primers amplified 4 to 10 bands ranging in size from 300 bp to 2000 bp, with an overall average of 6. 92 amplified bands per primer. Additionally, ISSR produced 1 cultivar-specific molecular marker. AMOVA(Analysis of molecular variance) software was used to calculate the Nei’s genetic distance and a dendrogram was constructed based on UPGMA cluster analysis. These 54 sweet osmanthus cultivars surveyed were classified into 7 major groups, and each cultivar in this study could be distinguished from others, suggesting that PCR-based ISSR analysis was an efficient method for cultivar identification. The efficiency of ISSR analysis for cultivar identification and classification of sweet olive were also discussed.