Gloriosa superba L. (Colchicaceae) is used in the treatment of gout and rheumatism as a traditional medicine dates back to 1810. It has also been used as ethnobotanical and folklore medicine to induce abortion/vaginal poison.The present study was carried out to identify the chemical variation existing in the major alkaloid metabolite (colchicine) in a threatened species, Gloriosa superba L. (Colchicaceae) and is correlated with invitro antigout activity.The samples (tuber) were collected from their natural locations in Gangetic plain of India. HPLC-PDA quantification of colchicine was done on C column at 245 nm and invitro antigout activity was analyzed by inhibition of protein denaturation, DPPH and Hydroxyl radical scavenging assay.The colchicine content within the 29 s ranges from 0.021 to 0.665% and the maximum contents was in NBG-10 from Kanth (U.P). Such high colchicine (0.665%) containing natural population of G. superba is reported for the first time in Indian population. Four chemotypes viz. NBG-10, NBG-120, NBG-126 and NBG-88 were selected on the basis of colchicine content for invitro antigout activity. NBG-10 was separated from rest of the population exhibiting the most promising activity with high colchicine content.The outcomes will be helpful in the identification of elite chemotype for herbal product development and quality check of metabolites in raw material. The study will also support the site-specific commercial cultivation to meet out the industrial demand as well as income generation to farmers.