Fast pyrolysis bio-oils are feasible energy carriers and a potential source of chemicals. Detailed characterization of bio-oils is essential to further develop its potential use. In this study, quantitative C-13 nuclear magnetic resonance (C-13 NMR) combined with comprehensive two-dimensional gas chromatography (GC x GC) was used to characterize fast pyrolysis bio-oils originated from pinewood, wheat straw, and rapeseed cake. The combination of both techniques provided new information on the chemical composition of bio-oils for further upgrading. C-13 NMR analysis indicated that pinewood-based bio-oil contained mostly methoxy/hydroxyl (approximate to 30%) and carbohydrate (approximate to 27%) carbons; wheat straw bio-oil showed to have high amount of alkyl (approximate to 35%) and aromatic (approximate to 30%) carbons, while rapeseed cake based bio-oil had great portions of alkyl carbons (approximate to 82%). More than 200 compounds were identified and quantified using GC x GC coupled to a flame ionization detector (FID) and a time of flight mass spectrometer (TOF-MS). Nonaromatics were the most abundant and comprised about 50% of the total mass of compounds identified and quantified via GC x GC. In addition, this analytical approach allowed the quantification of high value-added phenolic compounds, as well as of low molecular weight carboxylic acids and aldehydes, which exacerbate the unstable and corrosive character of the bio-oil.