An efficient micropropagation protocol for Rheum rhabarbarum L. was developed in this study. The in vitro rhubarb plants obtained in the multiplication stage (proliferation rate: 5.0 ± 0.5) were rooted in vitro (96% rooting percentage) and acclimatized ex vitro in floating perlite, with 90% acclimatization percentage. To assess the genetic fidelity between the mother plant and in vitro propagated plants, sequence-related amplified polymorphism (SRAP) markers were used. All banding profiles from the micropropagated plants were monomorphic and similar to those of the mother plant indicating 100% similarity. Regarding the polyphenolic profile, gallic, protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic, caffeic, syringic, p-coumaric and ferulic acid were present in different amounts (2.3-2690.3 μg g-1 dry plant), according to the extracted matrix. Aglicons and glycosides of different classes of flavonoids were also identified. The rhizome extracts (both from in vitro and field grown plants) contained resveratrol, a stilbene compound with high antioxidant properties, ranging between 229.4 to 371.7 μg g-1 plant. Our results suggest that in vitro propagation of Rheum rhabarbarum L. represents a reliable alternative to obtain a large number of true-to-type planting material with high bioactive compound content of this valuable nutritional and medicinal species.