One of the few similarities between petroleum and bio-oils derived from biomass pyrolysis is that they are both complex organic mixtures composed of thousands of distinct elemental compositions, but biomass pyrolysis oils uniquely contain ultrahigh oxygen content and a more diverse collection of chemical functionalities. Thus, their chemistry is different from fossil fuels, and advanced upgrading strategies for the coprocessing of such unique materials along with conventional refinery feeds will benefit from comprehensive knowledge of their molecular composition, known as petroleomics. The work presented herein focuses on the molecular characterization of nonvolatile species from a loblolly pine bio-oil and its hydrotreated effluents by soft ionization methods coupled to high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Electrospray ionization (ESI) facilitates the analysis of polar oxygen-containing molecules, whereas atmospheric pressure chemical ionization (APCI) enables access to hydrocarbons. The molecular data revealed time-dependent compositional changes, visualized in van Krevelen diagrams, that highlighted the optimal catalyst performance and the impacts of catalyst fouling or deactivation. Furthermore, elucidation of compositional features such as abundance-weighted H/C, O/C, molecular weight, and aromaticity facilitated data interpretation and suggested that value-added bio-oils are likely produced upon a concurrent decrease in oxygen content, aromaticity, and molecular weight with a marked increase in H/C. Furthermore, distinct temporal molecular changes suggested specific hydrotreatment reaction pathways, including concurrent deoxygenation and hydrogenation, transalkylation, and cracking of highly aromatic lignin-like oligomers. The detailed molecular characterization provided by FT-ICR MS facilitated access to common molecular formulas (those detected both before and after upgrading). Common formulas are hypothetically recalcitrant compounds, which feature a highly aromatic nature (low H/C) and alkyl deficiency. Understanding the chemistry of such molecules, along with the remaining oxygen-containing species, is critical for future advances in bio-oil upgrading.