Abstract Asphaltenes, the heaviest and most polar components of crudes, are generally associated with considerable operational issues in refineries. In order to understand potential operational issues during upgrading/processing of bio-crudes, structural and thermal behaviour of asphaltenes derived from bio-crude (bio-asphaltenes) from hydrothermal liquefaction of food-waste and wood residues were compared with petroleum derived asphaltenes derived from bitumen. Structural analysis using nuclear magnetic resonance and elemental analysis revealed 7 aromatic rings per unit structure for bitumen asphaltenes, 4 for food-waste asphaltenes, and 3 for wood asphaltenes. The calculated molecular weight per unit structure followed the order: bitumen asphaltenes (589–636 g mol−1) > food-waste derived asphaltenes (338–358 g mol−1)> wood residue derived asphaltenes (268–274 g mol−1). The carbon residues using thermal gravimetric analysis (bitumen asphaltenes = 40%, bio-asphaltenes = 19–25%) and glass transition temperature (bitumen asphaltenes = 80 °C, bio-asphaltenes = 4–64 °C) followed the same order. These results indicate a very different structural and thermal behaviour for petroleum and bio-asphaltenes.