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Olive oil is a fundamental component of the Mediterranean area diet and it is widely used as a condiment, cooking medium, emulsion component and in the storage of vegetable and animal food [1]. Olive oil cultivation originated in Asia Minor and has spread to Greece, Italy, Spain and North Africa. Recently it was also extended in other countries with temperate climate like France. In contrast to other vegetables oils, extra virgin olive oil (EVOO), produced by modern milling technologies, can be directly consumed without any further manipulation. Once a year, the International Olive Council (IOC) updates statistics on production, imports, exports and consumption of olive oil. The statistics of 2013 shows that EU is the responsible for 71.9% of world production of olive oil and Spain produces the 59.1% of Europe’s olive oil. Many areas where high-quality olive oils are produced are located in Spain. The characteristics of these oils are determined by a series of factors: the nature of the soil, the climate variety of the plant, cultivation and oil extraction techniques [2]. The Food Quality Certification are products that are protected by a European Union legislation ensuring compliance of higher requirements than those required for other products. Protected Designations of Origin (PDO), Protected Geographical Indications (PGI) and others are official EU classifications, and a system used in Spain for the recognition of distinct quality [3]. Only some works have been conducted on the identification of markers for the origin of oils by analytical methods [4–6]. Metabolomics (or more precisely metabolic fingerprinting) is a feasible approach for the discovering of those markers. In the Universidad Jaime I (UJI) some metabolomics projects have been carried out in the field of Food Authenticity. Very recently an article was published in Food Chemistry where some biomarkers were identified for correct authentication of Spanish (Valencian) oranges. In this project, 91 Extra Virgin Olive Oil (EVOO) samples from different regions of Spain with Protected Designation Origin (PDO) certificate have been analyzed using multivariate analysis from the data obtained by UHPLC-QTOF MS with the objective to discover useful biomarkers for origin differentiation. QTOF MS has been used under MSE mode, i.e. simultaneous acquisition at low (LE) and high collision energy (HE), which provides useful information on the molecular mass (commonly at LE) and on the main fragments ions (commonly at HE). The non-lipidic (unsaponifiable) and lipidic (saponifiable) portion of the oil (see in Figure 1 the olive oil composition) were analyzed separately because of the difference in polarity, that means, two separation methods were performed in UHPLC under reversed phase conditions but using different mobile phases. Furthermore, the samples were analyzed in both positive and negative electrospray ionization mode in the QTOF MS. This work only deals on the analysis of lipidic portion of the EVOO, composed mainly by triglycerides (a triglyceride is an ester derived from glycerol and three fatty acids). A freeware R-package has been used to extract and integrate peaks from the Total Ion Chromatogram (TIC), and to al techniques have been employed to classify samples according to their origin significant biomarkers have been highlighted in order different Spanish Regions.