Triacylglycerols (TAGs) 16:1(n-7)/16:1(n-7)/18:1(n-7) (Po/Po/V) and 16:1(n-7)/16:1(n-7)/18:1(n-9) (Po/Po/O) in pulp/peel oils of various sea buckthorn (Hippophaë rhamnoides) subspecies and varieties were separated by reversed-phase high-performance liquid chromatography. The regioisomerism of the TAGs was determined by tandem mass spectrometry using ammonia supplemented in the nebulizer gas to produce ammonium adducts. The regioisomer compositions of Po/Po/V (8−24% of PoVPo) and Po/Po/O (43−61% of PoOPo) both differed from the random distribution of fatty acids (33.3% of ABA) in all 32 sea buckthorn samples investigated. The regioisomer compositions were different between cultivated ssp. rhamnoides varieties, wild ssp. rhamnoides, and wild ssp. sinensis. Differences were also found in the regioisomerism of both Po/Po/V and Po/Po/O between the two cultivated ssp. rhamnoides varieties, Tytti and Terhi. In addition, growth location and harvesting years showed clear impacts on the regioisomer compositions of Po/Po/V and Po/Po/O. Higher temperatures showed positive correlations with the proportion of PoOPo in ssp. rhamnoides and wild ssp. sinensis and with the proportion of sn-PoPoV + sn-VPoPo in wild ssp. sinensis. However, higher temperatures, higher temperature sums, and radiation sums increased the accumulation of PoVPo in wild ssp. rhamnoides.