The critical challenge of hydrothermal liquefaction (HTL) for bio-oil production from biomass is the production of large amounts of aqueous products (HTL-AP) with high organic contents. The present study investigated the anaerobic digestion (AD) performances of HTL-AP under both thermophilic and mesophilic conditions, and molecular and metabolic analysis were conducted to provide insights into the different performances. The results showed that thermophilic AD had lower COD removal efficiency compared to mesophilic AD (45.0% vs. 61.6%). Liquid chromatography coupled with organic carbon detection and organic nitrogen (LC-OCD-OND) analysis showed that both high molecular weight (HMW) and low molecular weight (LMW) compounds were degraded to some extent and more LMW acids (LMWA) and recalcitrant aromatic compounds were degraded in the mesophilic reactor, which was the main reason of higher COD removal efficiency. Phenyl compounds (e.g. phenol and 2 methoxyphenol), furans and pyrazines were the recalcitrant chemicals detected through GC-MS analysis. Fourier transform ion cyclone resonance mass spectrometry (FT-ICR-MS) analysis demonstrated the complexity of HTL-AP and the proportions of phenolic or condensed aromatic compounds increased especially in the thermophilic effluents. Metabolites analysis showed that the reasons contributing to the differences of mesophilic and thermophilic AD were not only related to the degradation of organic compounds (e.g. benzoate degradation via CoA ligation) in HTL-AP but also related to the microbial autogenesis (e.g. fatty acid biosynthesis) as well as the environmental information processing. In addition, the enrichment of Mesotoga, responsible for the high degradation efficiency of LMWA, and Pelolinea, involved in the degradation of phenyl compounds, were found in mesophilic reactor, which was consistent with higher removal of corresponding organics.