SUMMARYDiosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16‐oxidations of cholesterol followed by a late C26‐oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16‐oxygenase(s) shows strict 22R‐stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S‐dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16‐oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R‐oxidation and DzCYP90G6 for the C16‐oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots.