Tire pyrolysis oil (TPO) is a complex mixture of hydrocarbons (HC), and it is one of the useful fractions obtained from the pyrolysis of waste tires (WT). As a result of its high energy density (HHV ∼43 MJ/kg), TPO use as a fuel in combustion systems is a promising approach for recycling WT. However, fundamental fuel characteristics and combustion properties of TPO are still unexplored, which stand as a bottleneck for potential applications. This work pursues a comprehensive understanding of the structural characteristics of a TPO produced in a lab-scale twin-auger reactor as a first step toward defining applications and upgrading strategies. Therefore, advanced analytical techniques such as Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy were utilized. In addition, we also present the characterization of a TPO obtained from adding CaO to WT, as a low-cost catalytic material for its in situ upgrading, herein named TPO[CaO]. FT-ICR MS results revealed the significant presence of pure HC (HC(TPO) = 74.9% and HC(TPO[CaO]) = 78.6%) and HC containing one sulfur atom (S1) (S1(TPO) = 14.3% and S1(TPO[CaO]) = 13.9%). HC compounds were found mainly in the form of triaromatics (26%), tetra-aromatics (13 and 15%), and penta-aromatics (22 and 30%), while S1 compounds were found mainly in the form of dibenzothiophene (31%) and benzonaphthothiophene (34%). The resolved compounds by means of FT-ICR MS exhibited an average double bond equivalent (DBE) number of 11.3 and 12.2 for TPO and TPO[CaO], respectively. These high DBE values were indicators of the significant presence of condensed aromatic structures. 1H NMR analysis showed that hydrogen atoms in methylene (CH2), methyl (CH3), and naphthenic groups and hydrogen atoms in aromatic structures make up more than 80% of both fuels. Similarly, carbon atoms in paraffinic groups (both CH2 and CH3) and protonated carbons in aromatic rings together form more than 50% of the carbon atoms in TPO and TPO[CaO]. The information reported in this work provides new insights into the structural characteristics of the TPO obtained in a promising technology as the twin-auger reactor for its use as a fuel, as well as for the design of upgrading strategies.