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 similar to 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 H-1 and C-13 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 (S-1) (S-1(TPO) = 14.3% and S-1(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. H-1 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.