The present study was conducted to study the genetic diversity among sex (6) olive cultivars and one unknown genotype grown in El-Kharga Oasis, El-Wadi El-Gedid, Egypt using inter-simple sequence repeats (ISSR) markers. Ten (ISSR) primers amplified 92.00 fragments of which 46.00 fragments were polymorphic. The number of polymorphic bands per primer varied from 0.00 to 8.00 with 4.60 bands per primer on average. Genetic similarities were calculated using the Dice coefficient (Nei and Li., 1979). The resulting similarity matrix was subjected to the UPGMA clustering method for dendrogram construction and cultivar differentiation. ISSRs dendrogram obtained by UPGMA analysis grouped the local cultivar and the unknown local genotype grown in El-Kharga, El-Wadi El-Gedid very close to each other, in the same time grouped all foreign cultivars near each other's and a part from the local ones. The results obtained could be used for selection the promising parents to generate an effective breeding program and for conservation strategies to future olive propagation program under the promising area El-Wadi El-Gedid conditions. Also, show that ISSR markers could be useful for cultivar differentiation and genetic diversity of Olea europaea L. studies.