TAG of seeds, berries, and fruit pulp/peel of different subspecies of sea buckthorn (Hippophaë rhamnoides) were analyzed by MS and tandem mass spectrometry (MS/MS). The seeds contained mainly TAG with acyl carbon number (ACN) of 52 with 2-6 double bonds (DB) (20-30%), and TAG of ACN 54 with 3-9 DB (70-80%). In the pulp/peel fraction, the major TAG were species with ACN:DB of 48:1 to 48:3 (19-49%), 50:1 to 50:4 (31-41%), and 52:1 to 52:6 (9-19%). The molecular weight species of whole berries largely resembled those of fruit pulp/peel with additional species of ACN 54 from the seeds (5-24%). Subspecies (ssp.) sinensis differed from ssp. mongolica and rhamnoides by having a higher proportion of TAG of ACN 52 (27% vs. 21% and 22%, P< 0.05) and a lower proportion of ACN 54 (71% vs. 79% and 78%, P < 0.01) in seed TAG. Seed TAG of ssp. mongolica contained a higher proportion of more unsaturated species compared with those of the two other subspecies. Berry TAG of ssp. mongolica had the highest proportion of molecular species of ACN 48 due to the higher proportion of palmitic and palmitoleic acids and the lower seed content of the berries. Overall, palmitic acid favored the sn-1 and sn-3 positions. The order of preference of unsaturated FA for the sn-2 position depended at least partially on the FA combination of TAG. Seed TAG of ssp. mongolica contained a higher proportion of alpha-linolenic acid in the sn-2 position than those of ssp. sinensis. In berry TAG, ssp. mongolica had the highest proportions of palmitoleic and linoleic acids in the sn-2 position, and the lowest proportion of oleic/cis-vaccenic acid in the sn-2 position, among the three subspecies.