Gentiana species belonging to the Gentianaceae family are medicinal plants rich in glycosides and phenolics. Gentiana lutea L. is a highly sought-after plant for medicinal and industrial purposes because of its content of secondary metabolites. Not only G. lutea, but also Gentiana boissieri Schott et Kotschy ex Boiss., another Gentiana species distributed only in Turkey, is on the endangered species list and is not allowed to be collected in more than 100 countries. For this reason, the development of an efficient in vitro culture method to obtain the secondary metabolites synthesized in its roots is important, as it allows the production of higher amounts of these valuable metabolites. This study was conducted to determine the appropriate inoculum density (ID, fresh root weight per unit volume to be used), which is an important factor for biomass and secondary metabolite production, for root growth and production of phenolics and glycosides in the root culture of G. lutea and G. boissieri. For this purpose, roots were cultured at different IDs (5, 10, 15, 20, 25, and 30 g L-1) and evaluated for fresh root weight, root growth index, root dry weight, and content of iridoid (loganic acid), secoiridoids (swertiamarin, sweroside, gentinopicroside), xanthan (isogentisin), total phenolic, and some important phenolic compounds. All parameters were found to vary depending on the IDs. The result of the study was that the highest values in both root growth and production of most metabolites were obtained from the roots of G. lutea and G. boissieri cultivated with 20 g L-1 and 5 g L-1 IDs, respectively.