The changes in total phenolics, flavonoids, tannins, valerenic acid, and antioxidant activity were assessed in 25 populations of Valeriana jatamansi sampled from 1200 to 2775 m asl and four habitat types of Uttarakhand, West Himalaya. Significant (p < 0.05) variations in total phenolics, flavonoids, valerenic acid, and antioxidant activity in aerial and root portions and across the populations were observed. Antioxidant activity measured by three in vitro antioxidant assays, i.e., 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic) (ABTS) radical scavenging, 2,2'-diphenyl-1-picryylhydrazyl (DPPH) free radical scavenging, and ferric-reducing antioxidant power (FRAP) assays, showed significant (p < 0.05) differences across the populations. However, no clear pattern was found in phytochemicals across the altitudinal range. Among habitat types, (pine, oak, mixed forest, and grassy land), variation in phytochemical content and antioxidant activity were observed. Equal class ranking, neighbor-joining cluster analysis, and principal component analysis (PCA) identified Talwari, Jaberkhet, Manjkhali, and Khirshu populations as promising sources with higher phytochemicals and antioxidant activity. The results recommended that the identified populations with higher value of phytochemicals and antioxidants can be utilized for mass multiplication and breeding program to meet the domestic as well as commercial demand.