Amplified fragment length polymorphism (AFLP) analysis was used to assess genetic inter-relationships among olive varieties cultivated in the Eastern Mediterranean Basin. The genotypes sampled included most of the important cultivars from Turkey, Greece and the Middle East and selected genotypes from the Western Mediterranean area. A total of 119 polymorphic markers were generated from five selective primer-pair combinations. The combined data sets generated by just two primer-pairs were adequate to discriminate between all 65 genotypes, while each primer-pair could individually identify up to 64 genotypes. A factorial correspondence analysis (FCA) plot indicated that the cultivars clustered into two relatively modestly defined groups. The first broad group was dominated by cultivars from Turkey but also included genotypes originating from the Middle East (Syria and Lebanon) that collectively formed a tight subcluster. The second group comprised Greek cultivars and those originating from the Western Mediterranean. A significant genetic distance value between Greek and Turkish cultivars was provided by an analysis of molecular variance (AMOVA). There was also evidence of substructure here, with an apparent separation of most Spanish and Italian clones. These findings are in general accordance to previous suggestions of an East-West divergence of olive cultivars, although the dichotomy is less extensive than reported previously and complicated by regional variation within each group.