In recent years, there is an increasing interest in high-quality extra virgin olive oils (EVOOs) produced from local cultivars. They have particular chemical/organoleptic characteristics and are frequently subjected to fraud, whereby the control of quality requires a powerful varietal check. In the present research, triacylglycerols (TAGs) and volatiles have been studied as chemical markers for the authentication of EVOO samples from four Italian varieties of Olea europea (Dolce Agogia, Frantoio, Leccino, and Moraiolo). The monocultivar EVOO samples have been subjected to a chemical⁻enzymatic chromatographic method in order to perform a stereospecific analysis, an important procedure for the characterization of TAG of food products. The results, combined with chemometric analysis (linear discriminant analysis, LDA), were elaborated in order to classify Italian EVOO monocultivar samples. In accordance with the total and intrapositional fatty acid (FA) composition of TAG fraction, the results were allowed to carry out a varietal discrimination. In addition, volatile compounds were also determined by solid-phase micro-extraction gas chromatography⁻mass spectrometry analysis. All EVOO samples were correctly classified when TAG stereospecific data and volatile results were elaborated by the LDA procedure, even if volatile compounds showed a higher discriminant power.