Tension and opposite wood, divided in the corresponding heartwood and sapwood regions, from 8-year old Eucalyptus globulus trees were characterized for chemical composition and fibre anatomy in the transverse section in order to identify the main differences. Thicker and poorly lignified cell walls were found in tension wood. Cellulose content was similar for both woods, although tension wood contained 30% higher xylose and 15% less lignin than opposite wood. Lignin of tension wood contained 24% more syringyl units and a higher frequency of β-O-4 linkages. Principal component analysis (PCA) performed separately with anatomical and chemical data showed a clear separation between tension and opposite wood. The principal characteristics used for separation were: cell wall thickness, vessel diameter, lumen diameter, lignin and xylose content, and amount of syringyl units.