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Low-lipid microalgae can be successfully converted to bio-crude oil in a hydrothermal liquefaction (HTL) environment. This study examined the behavior of hydrothermal liquefaction of two low-lipid content microalgae in subcritical water between 200 °C and 320 °C at 20 °C intervals. Under these conditions, the chemical composition and functional groups for the bio-crude oil and aqueous fraction were analyzed. Results indicated that reaction temperature greatly affected the distribution of chemical composition and functional groups of HTL bio-crude oil and aqueous fraction. The bio-crude oil with a higher percentage of aliphatic functional groups was obtained at higher reaction temperatures (280–320 °C). Besides, the aqueous fraction recovered under the same operating conditions had a lower concentration of nitrogenous organic compounds (NOCs) with two or more methyl groups. The general reaction network …