Use of wood feedstocks for sugar-based biorefineries requires suitable treatments of the various tree fractions to optimize yields. In the current study, stem wood fractions (sapwood, heartwood and knotwood) were sampled at different heights from well-documented Scots pine trees taken from two contrasting stands. The fractions were assessed in terms of chemical composition, response to SO2-catalysed steam pretreatment and enzymatic digestibility. There were significant differences in total extractive contents between the fractions, where the heartwood fractions had an extractive content 1-3 wt% higher than sapwood (corresponding to a relative increase of 20-60 %) for samples at the same height. In contrast, the differences in macromolecular carbohydrate contents between the fractions were smaller and mainly insignificant. One exception was the xylan content, which was higher in heartwood than in sapwood at the same tree height (a relative difference of 10-15 %). Steam pretreatment resulted in a clearly higher degree of hydrolysis for sapwood than for heartwood at the same conditions. However, at optimal pretreatment temperatures a higher total sugar yield was in fact obtained for heartwood, showing the importance of tuning the process conditions for the respective wood fractions.