6 α,7 α-Epoxy-5 α-hydroxy-1-oxo-22 R-witha-2,24-dienolide (CHO), 5 β,6 β-epoxy-4 β,20-dihydroxy-1-oxo-22 R-witha-2,24-dienolide (CHO), and 4 β,27-dihydroxy-1-oxo-22 R-witha-2,5,24-trienolide (CHO), were isolated from Withania somnifera roots. The structures of these withanolides were established by spectroscopic analysis and X-ray diffraction studies as withanolide B, withanolide D, and 5,6-deoxywithaferin A, respectively. All three compounds crystallize in the orthorhombic space group P222 with unit cell parameters: a = 9.1012(3) Å, b = 12.2728(4) Å, c = 21.5396(7) Å, Z = 4 for withanolide B; a = 7.6569(5) Å, b = 9.9426(7) Å, c = 33.767(2) Å, Z = 4 for withanolide D; and a = 6.9310(3) Å, b = 10.6745 (4) Å, c = 34.667(2) Å, Z = 4 for 5,6-deoxywithaferin A, respectively. The crystal structures have been solved by direct methods and refined to R = 0.024 for 4,290 unique reflections (withanolide B), R = 0.042 for 4,601 unique reflections (withanolide D), and R = 0.033 for 4,626 unique reflections (5,6-deoxywithaferin A), respectively. Rings B, C, D of the steroid skeletons are trans connected while ring C adopts a chair conformation, five-membered ring D adopts a half-chair, and the δ-lactone ring E in the side chain adopts a sofa conformation. For withanolide B, rings A and B are trans connected and both rings A and B exist in a half-chair conformation; for withanolide D, rings A and B are cis connected, and ring A has a sofa conformation while ring B has a distorted boat conformation; for 5,6-deoxywithaferin A, ring A has a half chair conformation and ring B exists as a boat conformation. Graphical Abstract: The crystal structures of 6 α,7 α-epoxy-5 α-hydroxy-1-oxo-22 R-witha-2,24-dienolide (withanolide B), 5 β,6 β-epoxy-4 β,20-dihydroxy-1-oxo-22 R-witha-2,24-dienolide (withanolide D), and 4 β,27-dihydroxy-1-oxo-22 R-witha-2,5,24-trienolide (5,6-deoxywithaferin A) are described.[Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]