Mushrooms are known to produce phytohormones, in particular cytokinins. Here we studied in vitro production of cytokinins in medicinal mushrooms. Cytokinins were identified and quantified in mycelial biomass of 13 species by using high-performance liquid chromatography-mass spectrometry. Trans-zeatin, zeatin riboside, zeatin-O-glucoside, isopentenyladenosine, and isopentenyladenine were found but only 1 species (Ganoderma lucidum) contained all these forms. Zeatin-type cytokinins predominated. Composition of the cytokinin pool was unique in each species. The largest total amount of cytokinins was detected in Morchella esculenta strain 1755 and the smallest amount, in Flammulina velutipes strain 1878. The productivity of cytokinin biosynthesis in mycelial biomass of mushrooms was the lowest in mycelial biomass of Sparassis crispa strain 314 and highest in Pleurotus ostreatus strain 551. F. velutipes strain 1878 and Cyclocybe aegerita strain 960 mycelial biomass showed the most productive zeatin riboside biosynthesis. We emphasize the need to take into account the biological activity of cytokinins, on the basis of the mycelial biomass of medicinal mushrooms, in the development of drugs or dietary supplements. Macromycetes with high rates of cytokinin biosynthesis are considered to be prospective producers of pharmacologically active compounds.