Inter-simple sequence repeat(ISSR) molecular markers were applied to analyze the genetic diversity and clustering of 48 introduced and bred cultivars of Olea euyopaea L. Totally 106 DNA bands were amplified by 11 screened primers, including 99 polymorphic bands; the percentage of polymorphic loci was 93.40%, indicating a rich genetic diversity in Olea euyopaea L. germplasm resources. Based on Nei's genetic distances between various cultivars, a dendrogram of 48 cultivars of Olea euyopaea L. was constructed using unweighted pair-group(UPMGA)method,which showed that 48 cultivars were clustered into four main categories; 84.6% of native cultivars were clustered into two categories; most of introduced cultivars were clustered based on their sources and main usages but not on their geographic origins. This study will provide references for the utilization and further genetic improvement of Olea euyopaea L. germplasm resources.