Collina di Brindisi is an Italian extra virgin olive oil that obtained the mark of protected designation of origin (PDO) according to EC Regulation 2081/92. The varietal requirements of the official production protocol of this oil foresee that this oil is prepared from cultivar Ogliarola (minimum 70%) and other Olea europaea L. cultivars that are diffused in the production area, accounting for a maximum of 30%. The aim of this work was to verify the effectiveness of microsatellite analysis in verifying the identity of Collina di Brindisi PDO olive oil. A preliminary assessment of product’s quality by means of chemical analyses was also carried out. Microsatellite analysis clarified that the generic name Ogliarola, indicated in the technical sheet of this PDO oil, actually corresponded to the Ogliarola salentina cultivar. Furthermore, the obtained results showed that the examination of a limited number of DNA microsatellites enables the identification of the Ogliarola salentina cultivar in this PDO oil.