Metabolite fingerprinting of Sitka spruce (Picea sitchensis) bark and sapwood was carried out by (1)H nuclear magnetic resonance after wounding and artificial inoculation with the white rot fungus Heterobasidion annosum sensu stricto. The aim was to determine whether metabolites would differ in clones showing differing levels of susceptibility to H. annosum, in the fungal as compared with the control treatment (wounding, no fungus) and the reference (healthy sample collected at 0 days), at two different locations on the host, and at different sampling times (3 and 43 days after treatment). The results suggested that different metabolic processes occur in bark and sapwood after wounding and fungal inoculation, compared with healthy samples collected before treatment: In bark, greater peaks were elicited in the aromatic region whereas, in sapwood, lower amounts of all metabolites were observed in inoculated samples, compared with healthy samples. Multivariate statistical analysis carried out with analysis of variance-principal component analysis showed highly significant effects of reference, location, and time (PC1), and significant effects of clone and fungus. Differences between clones were apparent in sapwood but not in bark and were due to peaks in the aliphatic and carbohydrate regions. Over time, in bark, there was a decrease in carbohydrate peaks, followed by an increase in aliphatic and aromatic peaks. Sapwood, by contrast, showed a decrease in all peaks, followed by an increase in carbohydrate and aliphatic peaks. Changes in carbohydrate levels were observed within the lesion compared with the more distal location in both bark and sapwood.