About 29 olive (Olea europaea L.) cultivars including oil and table olive cultivars originating from Tunisia and other Mediterranean countries, were genotyped using amplified fragment length polymorphism (AFLP) DNA markers. This technique is a rapid and efficient method for producing DNA fingerprints. Using nine AFLP primer combinations, we produced a total of 410 AFLP markers, among which 172 revealed polymorphism. The results demonstrated a high degree of polymorphism in the olive germplasm we examined with an average of 39%. These AFLP markers were analyzed to estimate genetic distances between pairs of cultivars using Jaccard's similarity coefficient. Furthermore, cluster and principal component analyses were performed in order to identify the genetic variation patterns. Two main groups were obtained: one comprising primarily small-fruited cultivars grown mainly for oil production and the other comprising large fruited cultivars (regardless of their end-use). Our results show no evidence of clustering of olive cultivars according to their geographic origin.