Sea buckthorn (Hippophaë rhamnoides) seeds, berries, and berry fractions are often used as sources of bioactive ingredients for health products. The aim of the present study was to analyze lignans in these fractions of sea buckthorn. Secoisolariciresinol and matairesinol in seeds, fruit pulp/peel, and whole berries of sea buckthorn of three subspecies were analyzed by isotope dilution gas chromatography-mass spectrometry. The total content of the two lignans secoisolariciresinol and matairesinol varied widely from 8 to 139 microg/100 g in fresh berries and from 51 to 319 microg/100 g in dry berries. The content of secoisolariciresinol varied in the range of 34-313 microg/100 g of dry mass in the fruit pulp/peel and 93-355 microg/100 g in dry seeds. The content of matairesinol fell within the range of 3-25 microg/100 g in dry pulp/peel and 1-13 microg/kg in dry seeds. Wild H. rhamnoides ssp. sinensis contained a significantly higher total level of secoisolariciresinol and matairesinol in dry seeds, dry berries, and fresh berries compared with wild ssp. rhamnoides (253 vs 135 microg/100 g, P < 0.01, in seeds; 224 vs 153 microg/100 g, P < 0.05, in dry berries; 71 vs 29 g/100 g, P < 0.01, in fresh berries) and the cultivar of ssp. mongolica (253 vs 112 microg/100 g in seeds, 71 vs 9 microg/100 g in fresh berries). Harvesting dates had a significant influence on the content of the two lignans in seeds, fruit pulp/peel, and whole berries. This is the first report of lignans in sea buckthorn.