Separation of 1,2(2,3)- and 1,3-positional isomers of diacylglycerols (DAG) from vegetable oils by reversed-phase high-performance liquid chromatography (RP-HPLC) is investigated. The method is based on isocratic elution using 100% acetonitrile and UV detection at 205 nm. The following elution order of DAG molecular species is identified: 1,3-dilinolein < 1,2-dilinolein < 1,3-dimyristin < 1-oleoyl-3-linoleoyl-glycerol < 1,2-dimyristoyl-rac-glycerol < 1(2)-oleoyl-2(3)-linoleoyl-glycerol < 1-linolenoyl-3-stearoyl-glycerol < 1(2)-linolenoyl-2(3)-stearoyl-glycerol < 1,3-diolein < 1-palmitoyl-3-oleoyl-glycerol < 1,2-dioleoyl-sn-glycerol < 1(2)-palmitoyl-2(3)-oleoyl-glycerol < 1-linoleoyl-3-stearoyl-glycerol < 1,3-dipalmitin < 1(2)-linoleoyl-2(3)-stearoyl-glycerol < 1-oleoyl-3-stearoyl-glycerol < 1,2-dipalmitoyl-rac-glycerol < 1-palmitoyl-3-stearoyl-sn-glycerol < 1,3-distearin < 1,2-distearoyl-rac-glycerol. Linearity is observed over three orders of magnitude. Limits of detection and quantitation range 0.2-0.7 microg/mL for 1,3-dilinolein to 0.6-1.9 microg/mL for 1,2-dioleoyl-sn-glycerol, respectively. Precision and accuracy of the method are also demonstrated. The method is developed to separate mixtures of DAG molecular species produced from edible oils.