Extraction of hemicelluloses with subcritical water is an efficient and environmentally benign way of fractionating biomass, including lignocelluloses. The composition and extraction behavior of hemicelluloses from wood depends significantly on the species, which affects the obtained product and the requirements of the extraction process. The current work focuses on the extraction of hemicelluloses from three chemically and structurally different, interesting and economically important tree species namely stone pine (Pinta pinea), holm oak (Quercus ilex), and Norway spruce (Picea abies), for which the literature on holm oak and stone pine hemicelluloses is almost non-existent. The work was performed with identical experimental methods, to obtain completely comparable results. The effect of temperature, raw material and solid-liquid ratio on the extraction kinetics, pH and molar mass, was studied. The results revealed significant differences in the extraction behavior and composition of the different species.