Out of 814 threatened plants of India, Picrorhiza scrophulariiflora Pennell (Scrophulariaceae) is one such threatened species of Indian Himalayan region, having medicinal properties. Even though its existence is in threat due to anthropogenic as well as some other ecological factors, yet there is no any report on proper conservation measures. To check the exact current population status of both the species, the selected populations were analyzed using vertical belt transact model, which revealed that the P. scrophulariiflora is vulnerable in Sikkim Himalayas. Therefore, in order to exploit this high valued species for mass cultivation, simple and highly reproducible protocols for micropropagation, somatic embryos, direct shoot regeneration from callus and production of synthetic seed have been developed after selecting elite line and for this purpose picroside I and picroside II were used as marker molecules. The HPLC analysis revealed that the population of P. scrophulariiflora from Ha, Bhutan contains highest total picroside (i.e., picroside I and picroside II) with a total of 7.33% (picroside I= 2.21 and picroside II= 5.12% respectively). Therefore, the population of Ha, Bhutan was considered as elite line. The morphological and biochemical characters of the studied populations varied exclusively in terms of soil characters as well altitudinal gradients; therefore, attempt has also been made to correlate those soil and altitudinal parameters with the biomass of the species. To check whether these variations in active biomolecules content and biomass of this medicinal plant is due to those environmental factors or due to genetic factors, genetic diversity studies were undertaken using RAPD markers. The dendrogram clustered the individual of P. scrophulariiflora into 4 sub-groups according to the geographical regions indicating the variations in total picroside content may be due to genetic factors rather than the altitude or other environmental factors.