Picrorhiza kurrooa Royle ex Benth. is one of the well-established herbal plants with an exceptional therapeutic potential. It belongs to the Scrophulariaceae family and is commonly called as kutki. The drug obtained from the plant is a bitter tonic due to the presence of kutkin in it. Over 61 secondary metabolites from the plants have been identified, including iridoid glycosides, flavonoids, cucurbitacins, and phenolic chemicals. However, picrosides are the major phytochemicals in this species that are responsible for its well-known hepatoprotective properties. The present study was conducted to compare Picrorhiza kurrooa (dried rhizomes) obtained from local traders from the markets of three different districts of Uttarakhand, i.e., the Dewal block of Chamoli, Ukhimath block of Rudraprayag, and Dharchula block of Pithoragarh. Biochemical analysis was conducted on the powder of dried rhizomes for alkaloids, phenolics, tannins, flavonoids, and antioxidant activity. Based on analysis, it was found that the total phenolic content, total flavonoid content, total alkaloid content, and radical scavenging activity of P. kurrooa rhizomes purchased from Darma valley, Pithoragarh district of Uttarakhand, were the highest, followed by rhizomes collected from the Dewal block of Chamoli district and the least were found in rhizomes obtained from the Ukhimath block of Rudraprayag district of Uttarakhand. The maximum tannin content was found in Picrorhiza kurrooa rhizomes obtained from the Dewal block of Chamoli, while total reducing power was observed the highest in rhizomes from the Ukhimath block of Rudraprayag. The results provided evidence that P. kurrooa obtained from Darma valley, Pithoragarh, are the potential source of phenolics, flavonoids, and tannins and have the highest DPPH-scavenging activity and therefore could be served as the basis for future drugs and food materials.