In recent years, olive varieties started being cultivated in Brazil and the first genuine Brazilian virgin olive oils (VOOs) were produced. Even though some brands of Brazilian VOOs can already be found in the supermarkets, there is yet a lack of data about their chemical composition. The aim of this work was to determine the tocopherols, phytosterols and triterpenic alcohols contents of 25 VOOs produced from different olive varieties cultivated in three Brazilian states, Rio Grande do Sul, Santa Catarina and Minas Gerais, in two different crop years. The extraction protocol consisted in a procedure coupling a hot saponification step to liquid-liquid extraction. High-performance liquid chromatography coupled to ion trap tandem mass spectrometry (HPLC-APCI-IT-MS/MS) was used as analytical technique for the compounds identification and quantitation. Compounds separation was achieved using an AccelaTM U-HPLC liquid chromatography system with a C18 reversed-phase column and water (solvent A) and acetonitrile (solvent B), both acidified with 0.1% acetic acid, as mobile phase. Separated compounds were directed to a LCQ Fleet 3-D Trap mass spectrometer with an atmospheric pressure chemical ionization (APCI) source in positive ion mode and performing MS and MS/MS experiments. The HPLC-APCI-IT-MS/MS method used for phytosterols, tocopherols and triterpene alcohols analysis was validated for limits of detection and quantification, intra- and inter-day instrumental precision, and linearity range. This study was the first to characterize the unsaponifiable fraction of Brazilian VOOs. The main compounds detected were lupeol (0.29 - 3.38 mg/kg), β-amyrin (6.85 - 40.85 mg/kg), Δ5-avenasterol (42.95 - 578.99 mg/kg), stigmasterol (0.88 - 35.61 mg/kg), sitostanol (0.29 - 12.86 mg/kg), β-sitosterol (309.49 - 964.76 mg/kg), α-tocopherol (8.77 - 124.05 mg/kg), and β- + γ-tocopherol (0.67 - 12.18 mg/kg), matching those observed for well-established VOO from the countries with high experience in their production and indicating that VOO production in Brazil is promising.