A method for stereocontrolled chemical synthesis of P‐substituted nucleoside 5′‐O‐phosphorothioates has been elaborated. Selected 3′‐O‐acylated deoxyribonucleoside‐ and 2′,3′‐O,O‐diacylated ribonucleoside‐5′‐O‐(2‐thio‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s were chromatographically separated into P‐diastereomers. Their reaction with anions of phosphorus‐containing acids was highly stereoselective (≥90%) and furnished corresponding P‐chiral α‐thiodiphosphates and their phosphonate analogs with satisfactory yield. Chirality, 2011. © 2010 Wiley‐Liss, Inc.