Culinary rhubarb is grown for its large, thick leaf petioles. Red-stalked cultivars and selection are more attractive for the fresh market and processing industry. In vitro cultures are important for rapidly multiplying value genotypes and producing plants free of viruses. This study aimed to develop an in vitro propagation method for six rhubarb selections from the Raspberry (R1, R2, R3), Leader (L1, L2), and Karpow Lipskiego (KL) groups. In addition, the planting material of six rhubarb selections was subjected to phytochemical analysis using the HPLC method to assess the content of bioactive compounds. The cultures were initiated from underground buds on the crowns. The initial growth was obtained for 45 to 75% of initial explants because of bacterial contamination and low bud activity of some genotypes. The type and concentration of cytokinin and its interaction with gibberellin acid (GA3) were shown to have a significant effect on the cyclic multiplication and commercially interesting rate of all rhubarb genotypes. A high in vitro rooting frequency, 93.7 to 95.8% for rhubarb Raspberry, 94.3 to 100% for rhubarb Leader, and 96.7% KP selections were obtained after two-cycle rooting on a medium containing IBA and NAA. The polyphenolic compounds, such as cyanidin-3-O-rutinoside, rhaponticin, resveratrol, caffeic acid, p-coumaric acid, cinnamic acid, syringic acid, and ferulic acid were detected in selected rhubarb genotypes. The highest content of anthocyanins (2.9 mg·1 g−1 DM) and rhaponticin (107.8 µg·1 g−1 DM) was found in Raspberry selections. On the other hand, Leader selections were characterized by the highest content of resveratrol (0.25 µg·1 g−1 DM) and phenolic acids (1.3 µg·1 g−1 DM). The less attractive for functional food production seems to be KL selection.