Pyrolysis bio-oil is a liquid product of the pyrolysis of biomass. After appropriate upgrading, it can be used as biofuel or as a source of valuable chemicals. To optimize a pyrolysis process, a detailed understanding of the chemical composition of bio-oils is necessary. Nonvolatile and high-molecular-weight bio-oil components have usually been analyzed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers that provide the greatest mass resolution and mass accuracy among all currently available high-resolution mass spectrometers. Nevertheless, the main drawbacks of FT-ICR mass spectrometry are very high purchase and operating costs, which limit the use of this method for routine applications. Among other high-resolution mass spectrometers, the orbitrap mass spectrometer is the most promising candidate for being used as an alternative to FT-ICR for analyzing complex samples. In this work, we used negative-ion orbitrap mass spectrometry to characterize nonvolatile and high-molecular-weight components in the wood-derived fast pyrolysis bio-oil. We wondered which level of the mass resolution is necessary for the routine orbitrap mass spectrometry characterization of pyrolysis bio-oil from lignocellulosic biomass. Thus, we applied two different orbitrap mass spectrometers for the bio-oil analysis: LTQ Orbitrap Velos and LTQ Orbitrap Elite. By both instruments, mass spectra were acquired at different mass resolution settings (30 000 and 100 000 at m/z 400 by Orbitrap Velos and 240 000 and 480 000 at m/z 400 by Orbitrap Elite), and the obtained results were compared. To achieve the comprehensive characterization, we combined negative-ion electrospray–mass spectrometry (ESI–MS) and negative-ion atmospheric pressure chemical ionization–mass spectrometry (APCI–MS). We identified around 1900 bio-oil components. We observed no differences in the results obtained by the Orbitrap Elite at different mass resolutions and only minor differences between the results obtained by both mass spectrometers. The obtained results demonstrate the suitability of Orbitrap Elite and Orbitrap Velos (operating in the negative-ion mode) for the routine characterization of pyrolysis bio-oils from lignocellulosic biomass.