Lignan concentrations in Norway spruce (Picea abies [L.] Karst.) knotwood were studied in two long-term nitrogen fertilised experimental sites: a boreal site (66°5′N, 27°07′E) close to the Arctic tree line in northern Finland and a boreal site (61°10′N, 26°03′E) in southern Finland. Altogether 20 mature trees, representing five different size classes, were felled in the autumn 2002 and knots of the largest and smallest diameter in each whorl at the height of 4 m were analysed. Within-stem variation of knotwood lignans was studied at the 4 m intervals from stump height to the top from five control trees in the southern site. The following knotwood lignans were identified with GC-MS: hydroxymatairesinol (two isomers), secoisolariciresinol, α-conidendrin, α-conidendric acid, isohydroxymatairesinol, lariciresinol, lignan A, matairesinol, nortrachelogenin, todolactol A and isoliovil. The predominant lignan in Norway spruce knotwood was hydroxymatairesinol comprising approximately 77% of the total concentration of lignans. In mature trees in the southern site, the lignan concentrations were highest close to the starting point of the living branches. In the northern site, the total concentration of knotwood lignans was significantly higher (approximately 14% of the dry mass) than in the southern site (approximately 5.4% of the dry mass). In the northern site in control trees, a negative linear correlation was detected between branch diameter and hydroxymatairesinol concentration, but the similar correlation was not detected in fertilised trees or in the trees of the southern site. The possible reasons for higher hydroxymatairesinol concentrations in the northern knots were discussed.