When we research the genetic diversity of olives, we have to keep in mind the existence of three types of olives: wild olives, feral forms and cultivated olives. In this study, the samples of three wild olive populations from the islands of Pag and Lastovo, the peninsula Pelješac and 15 most common Croatian cultivars were collected. An analysis of 12 microsatellite loci was performed. Various genetic parameters were calculated and different statistical methods were used such as: polymorphism information content, average number of alleles per population, allelic richness, number of private alleles, observed and expected heterozygosity, inbreeding coefficient, genetic differentiation, calculation of genetic distances, creation of NJ tree, Bayesian model-based cluster analysis and factorial correspondence analysis. The individuals of the population Pelješac were characterized as feral forms not as true wild olives (oleaster). Wild origin has only been confirmed to the populations Pag and Lastovo. The cultivars Piculja and Mrčakinja were not cultivars but true wild olives, which, due to their abundant and regular flowering, serve as an abundant source of pollen during pollination season in the olive groves. The significant FIS coefficient in the Pag population indicates that this wild population, probably due to a decrease in size, has undergone a genetic bottleneck. The obtained results underlined the need to conserve natural populations of wild olives as reserves of rare and valuable alleles in breeding programs.