This study was carried out to understand the effect of MeJA on root growth, in vitro production of some bioactive compounds, and expression levels of the 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), secologanin synthase (SLS), 3-hydroxy-3-methylglutaryl-CoA (HMGR), and geranyl diphosphate synthase (GPPS) genes in root cultures of endangered Gentiana lutea and endemic Gentiana boissieri. For this purpose, MeJA at different concentrations (0.1, 0.5, and 1.0 mM) was applied to the one-week-old root cultures for 2, 4, 6, 8, and 10 days. After harvest, roots were evaluated in terms of root growth criteria and contents of loganic acid (iridoid), isogentisin (xanthone), swertiamarin, sweroside, amarogentin, and gentiopicroside (secoiridoids). In the study, the effects of MeJA applications on the activities of the DXR, SLS, HMGR, and GPPS genes, believed to be responsible for iridoid, xanthone, and secoiridoid synthesis, were also investigated. It was determined that all the criteria examined significantly changed depending on MeJA concentration and harvest time. Regarding metabolite contents, the most appropriate applications were found to be 0.5 mM and 1.0 mM MeJA for 8 days for G. lutea and 0.5 mM MeJA for 4 days for G. boissieri. Additionally, the expression levels of the genes were found to change with MeJA treatments, but it was noted that establishing a linear relationship with a single substance was not possible. Another noteworthy result was that the accumulation of loganic acid, swertiamarin, sweroside, and gentiopicroside was higher in the roots of G. boissieri than in those of G. lutea. In conclusion, it was determined that in vitro production of iridoid, xanthone, and secoiridoids with significant pharmaceutical effects in Gentiana root cultures could be increased with MeJA applications. The study also revealed the need for more detailed research, including genomic analyses of Gentiana species and identification of genes associated with metabolite biosynthesis.