In the Mediterranean basin, a large number of olive varieties are present. This poses a series of problems concerning germplasm characterization and management. In addition, there is a problem arising from the existence of homonyms and synonyms. This makes cultivar identification very difficult and complex. Microsatellites or simple sequence repeat (SSR) are locus-specific codominant markers showing a high degree of polymorphism and multiple alleles per locus. Their high informativeness makes them the markers of choice in genetic diversity studies. This work presents the results of molecular characterization and identification of 20 Tunisian olive varieties using 10 SSR markers. All the SSR amplification products were sequenced to determine the number of repeats and the range of allele size. The number of alleles per SSR varied from three to six and the average heterozygosity rate ranged from 30% to 95%. Hierarchical classification of varieties base on similarity measures and clustering was globally consistent with the grouping of varieties by end use and phenotypic characteristics. The result is that varieties having the same name were found to have a clonal relationship. Paternity analysis showed also clone relationships between varieties not known to be related.