Metolachlor is a chiral herbicide consisting of four stereoisomers, which is typically used as a racemic mixture or is enriched with the herbicidally active 1'S-isomers. Because studies on the enantioselective behavior of phyto-biochemical processes and the environmental fate of metolachlor have become significant, a practical method for analyzing and separating metolachlor stereoisomers must be developed. In the present study, the enantiomeric separation of metolachlor was achieved using OD-H, AS-H, OJ-H and AY-H chiral columns. The effects of different organic modifiers in an n-hexane-based mobile phase were investigated, and various temperatures and flow rates, which may influence metolachlor separation, were also explored. The optimal resolution was obtained using an AY-H column with n-hexane/EtOH (96/4) as the mobile phase at a rate and temperature of 0.6mLmin(-1) and 25°C, respectively. The absolute configuration of the four stereoisomers was identified as αSS, αRS, αSR, αRR using computed and experimentally measured ECD and VCD spectra. Thermal interconversion and solvent stability experiments were also performed. Pure metolachlor stereoisomers in different organic solvents and water at 4°C or 30°C were stable. These results were used to establish a sound method for analyzing, preparing, characterizing, and preserving individual metolachlor stereoisomers in most natural environments.