Abstract Oligolignans present in substantial amounts in Norway spruce and Scots pine knots were characterised. The hydrophilic knotwood substances were extracted and different chromatographic methods were applied to obtain fractions for analysis by GC, GC-MS, HR-EI-MS, LC-ESI-MS, NMR, and FTIR. β-O-4-Linked guaiacylglyceryl ethers of hydroxymatairesinol (Ia, Ib, Ic), secoisolariciresinol (II), lariciresinol (III), isolariciresinol (IV), lignan A (V), liovil (VI), conidendrin (VII), and pinoresinol (VIII) were identified in the spruce knotwood extract and β-O-4-linked guaiacylglyceryl ethers of nortrachelogenin (IX) and secoisolariciresinol (II) in the pine knotwood extract. The structures of allo-hydroxymatairesinol-4′-guaiacylglyceryl ether (Ia), hydroxymatairesinol-4-guaiacylglyceryl ether (Ib), hydroxymatairesinol-4′-guaiacylglyceryl ether (Ic), secoisolariciresisinol-4-guaiacylglyceryl ether (II), and lariciresinol-4′-guaiacylglyceryl ether (III) were determined via GC-MS, HR-MS, and 1H and 13C NMR analyses. The structures of (Ia), (Ic), (IV), (V), (VI), (VII), and (IX) have not been reported earlier. Spruce stemwood, both heartwood and sapwood, also contained oligolignans in small amounts. In addition, some dilignans with four phenylpropanoid units were tentatively identified in the hydrophilic knotwood substances.