Rhodiola rosea L. is an endangered medicinal plant distributed in mountains and in high latitude regions. For its conservation, sustainable methods for the obtaining of its bioactive compounds must be developed. This work hypothesized that leaf, stem and rhizome explants of R. rosea from different geographical origins respond differently to inoculation with Rhizobium rhizogenes agropine strain ATCC43057. The objective was to generate R. rosea hairy roots (HRs) containing rol-genes. These HRs could be cultivated under axenic conditions for the extraction of the medical compounds rosavinoids and salidroside. Hereby, production of bioactive compounds could be improved per plant biomass. Thirteen R. rosea accessions of Alpine, Scandinavian, Nordic Gene Bank (NGB) and Russian origins were compared for their explant survival and HR formation. Significant differences were observed among plants from different geographical origins, where the NGB leaf explants exhibited up to 70% of HR formation and the Russian accessions did not exhibit HRs at all. Moreover, maintaining explants in light conditions after R. rhizogenes inoculation resulted in higher explant survival and HR formation rate (35%) when compared with explants kept in darkness (9%). Taken together, an efficient HR formation in roseroot by inoculation of R. rhizogenes following culturing in light was reported as a required step. This work represents a stepping-stone to R. rosea HR cultivation in bioreactors as well as regenerating whole plants. Hence, it is initiating a novel route towards high-throughput production of bioactive compounds as well preventing depletion of natural roseroot populations.