Posaconazole represents a triazole antifungal agent which is used to treat various fungal infections. It contains four chiral centers leading to 16 stereoisomers. With the convergent synthesis route, only 11 related stereoisomeric impurities may potentially exist in the active pharmaceutical ingredient (API). It is a challenge to separate all the stereoisomers in one run. To address this problem, a multiple heart-cutting chiral-chiral two-dimensional liquid chromatography (2D-LC) method was developed. The multiple heart-cutting 2D-LC separation was implemented on 2D-LC system with three chiral columns with immobilized polysaccharide chiral stationary phases, namely Chiralpak IB, IC and IF3. In the system, the column Chiralpak IB was used as the D separation column and IC and IF3 columns were used parallelly for the D separation. The twelve stereoisomers were all well separated in one run by the multiple heart-cutting chiral-chiral 2D-LC system.