Hydrothermal liquefaction of biomass produces a complex biocrude, which can be further upgraded to biofuel or chemicals, but there is a need for improved molecular understanding of product composition and reaction pathways. This study extensively characterizes semivolatile compounds in biocrudes from hydrothermal liquefaction (HTL) of microalgae (N. gaditana, C. vulgaris), macroalgae hyperborea), residue (dried distillers grains with solubles), and lignocellulosic (M. X giganteus). The biocrudes were analyzed using 2D gas chromatography coupled to time-of-flight mass spectrometry with in situ silylation. A total of 73 fatty acids were detected of which C-18 and C-20 compounds were most diverse, while palmitic acid was the single most abundant fatty acid. Multiple fatty acid amides were detected in biocrude from N. gaditana while being almost absent in samples from other lipid- and protein-containing feedstocks. Several alkylated indoles and quinolines were observed in biocrudes from protein-containing feedstocks. Monoglycerides, indanones, and alkylated benzenediols and chromen-2-ones, which are typically not reported, were also detected. These results provide new knowledge of a biocrude fraction, which is difficult to characterize.