Accurate identification of olive cultivars is an essential requirement for the oliveculture. Distribution of incorrectly labelled Olea europaea and the global spread of vegetative propagated cuttings over hundreds of years sometimes changed the names of the same cultivar growing in different regions. Development of cultivar specific markers is necessary for cultivars identification and protection and cultivar purity determination. Standard DNA marker types and universal database integrated with exiting agro-morphological identification could have a significant impact on the exact cultivar identification of olive plants. The putative synonymous Olea europaea cultivars Arancino, Carboncella, Corniolo, Moraiolo and Tondello have been characterised by different molecular methodologies. Cucca is another cultivar used for comparison. Analyses for olive cultivar identification were carried out through PCR amplification, restriction and cloning of spacers sequences of the nuclear ribosomal gene. The internal transcribed spacers region (ITS1, 5.8S and ITS2), was amplified by the polymerase chain reaction (PCR) by using two primers complementary to the 3’ region of 18S and 5’ region of 25S rDNA. The PCR product amplified from Moraiolo cultivar appears as a single band of approximately 700 bp in size: it was cloned using the pCR 2.1 vector. The ITS1 (about 250 bp), ITS2 (about 200 bp) and 5.8S rRNA gene were completely sequenced. No amplified length polymorphism was found among the analysed cultivars. The nuclear ribosomal intergenic spacer (IGS) and the flanking 5’ external transcribed spacer were amplified by using primers matching 3’ region of 25S and 5’ region of 18S. A partial sequence from Arancino cultivar was obtained. Amplicon restriction analyses were used to evaluate the genetic diversity among cultivars: no differential polymorphism was detected among them.