The objective of this study was to investigate the variation in internal knot size of Scots pine (Pinus sylvestris L.) stems sampled from mature permanent plots, and for which the silvicultural history was known. It was based on a sample of mature trees removed from two different spacing trials representing a moderate and high site index. Knot size was measured with non-destructive methods using a CT-scanner and digital image analysis. Initial spacing varied between 0.75 and 3 m on the high site-index trial and between 1.5 and 2.5 m on the moderate site-index trial. Wider initial spacing on the high site index resulted in larger knots near the base of the stem. However, due to successive thinnings which gradually equalised stand density among plots, the difference between most plots was less further up in the stems. The effect of silvicultural regime was much more limited on the lower site index. Within-stand differentiation resulted in a variation of tree diameter (DBH); larger trees had significantly larger knots. Furthermore, knots were larger towards south than towards north in both trials. These results illustrated that, by using non-destructive measurements on trees sampled from permanent research plots, it was possible to simultaneously study the variation of internal knot size at stand (such as site and silviculture effects), within-stand (such as relative tree size) and within-tree levels (such as height and azimuth). However, lack of replication prevented valid statistical inference as to stand-level effects.