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Next-generation biofuels have been widely investigated because they have particular advantages compared to firstgeneration biofuels. Pyrolysis is an example of a thermochemical route extensively used in oil and coal industries worldwide to produce these biofuels. Strategies for low-cost upgrading are among the biggest challenges facing the adoption of bio-oils in the development of commercial biofuels. Specific biomass sources could be the best option for generating bio-oil with the required properties. For this, it is necessary to understand the composition of these biomasses and their bio-oils. Here, we analyzed bio-oil samples from the fast pyrolysis of different biomasses collected during two different steps of the process by direct-infusion highresolution mass spectrometry. First, a comparative study of two common high-resolution mass spectrometers, quadrupole timeof-flight mass spectrometry (Q-TOF MS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), was performed to validate the methodology and to investigate the differences in mass discrimination and resolution. FT-ICR MS showed the best performance because of its unsurpassed resolution and accuracy. We apply the common petroleomics tools to interpret the mass spectra obtained. The FT-ICR MS analysis reveals that bio-oils are dominated by O x species. The class profile of bio-oils was strongly affected by the biomass and steps of the pyrolysis process.