Up to now, various categories of DNA-based markers have been employed in Olea europaea L. with cultivar identification purposes but none has been effectively used to analyse the olive oils. Since generally microsatellites have short sizes, this enables expecting good possibilities of detection also in the case of degraded DNA such as that recovered from olive oil. In this work, we aimed to apply the analysis of these markers to virgin oils obtained from five different olive cultivars, with fingerprinting purposes. Certified plant material was used and the oil was extracted from drupes by means of a laboratory-scale plant. Then, after oil centrifugation and DNA extraction from the remaining cellular residue, the amplification reactions were carried out and ten primers were tested. Out of them, four primers were chosen to achieve fingerprinting of the five types of oil and a multiplex DNA amplification was set up, basing on two of them, to obtain more information on a unique PCR reaction.