Palm oil (PO) mainly constitutes of 90-98% of triacylglycerol, 2-6% of diacylglycerol (DAG) and 2-5% of monoacylglycerol. This study was carried out to produce PO that is high in DAG through direct esterification using 1,3 positional specific lipase from Rhizomucor miehei (RM IM) and Thermomyces lanuginose (TL IM) as catalysts. Palm olein oil has been hydrolysed by water and 4% enzyme in a controlled water bath at 300 rpm for 48 hours at a temperature of 60±2oC while the esterification process was carried out in a same condition except it was run for 24 hours only. Samples DAG A1 and DAG B1 were synthesized products of the first replication using 10% enzyme TL IM and 10% enzyme RM IM respectively while samples DAG A2 and DAG B2 were synthesized products of second replication. DAG spots found on the thin layer chromatography (TLC) plates of samples DAG A1, A2, B1 and B2 bigger than the spot of the control sample. Based on high performance liquid chromatography chromatogram peak area, the total DAG accumulation showed significant differences (p<0.05) between the usage of enzymes TL IM and RM IM which were 34.28% and 45.67% respectively. The esterification method has clearly increased the DAG content of the control sample which was only 3.17%. Significant differences (p<0.05) also existed in the iodine value (IV), melting and crystallization temperature of all the samples. IV of control sample, DAG A and B were respectively 56.00, 35.00 and 30.50. Differential scanning calorimetry curves showed the melting and crystallization temperature were respectively -3.73oC and -5.72oC for samples using TL IM while -4.92 and -6.56 respectively for RM IM. The results concluded that the usage of enzyme RM IM is more effective in the production of PO high in DAG and efficiency of direct esterification process has been proved.