Cannabis sativa L. is a valuable plant that has regained its importance for medicinal use. Gas chromatography-mass spectrometry based untargeted metabolomic study was conducted in seeds of two accessions from different environments. A total of 236 metabolites were observed, and 43 metabolites were found differentially significant (p ≤ 0.05) in both the accessions. Based on the qualitative and quantitative accumulation of the nutraceutically important amino acids, cannabinoids, alkaloids, and fatty acids, the high altitude temperate Himalayan accession (CAN2) was found to have an advantage over the low altitude subtropical accession (CAN1). Seed oil from CAN2 showed the exclusive presence of linoleic acid and α- linolenic acid. The reducing power and DNA nicking assay on the methanolic extracts suggested higher antioxidant and nutraceutical potential in CAN2 and corroborated with the metabolic content of phenols and flavonoids. The environmental effect on the antioxidant and nutraceutical value in seeds is further discussed. © 2020 Elsevier Ltd