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Olea europaea L. is a particularly long-lived tree species characteristic of the Mediterranean region. In this study 44 standard cultivars and 89 wild trees derived from sexual reproduction in different geographic origins were scored for seven leaf enzyme systems. Genetic analyses showed that nine of 11 studied loci were polymorphic, with an average 2.7 alleles per locus in all trees analyzed. Wild trees had, on average, 66% heterozygous loci, and cultivated trees had only 19% heterozygous loci. Moreover, when they grew together, the wild trees, which are probably the result of interpollination between extant and/or ancient cultivars, possessed higher allele diversity than their sympatric cultivars. Thirty-six distinct multilocus genotypes allowed the identification of 75% of the standard cultivars. A significant positive association was found between vigor and heterozygosity at the EST1 locus in cross progeny, and at the EST1 and LAP1 loci for trees from natural populations. Despite its early and intensive domestication, the O. europaea L. thus shows a high level of enzyme polymorphism with the occurrence of novel genotype combinations in wild material