Technical Abstract: Soy-polyol oils (oxygenated acylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. Currently, they are produced by a two-step chemical process involving epoxidation and then the subsequent opening of the oxirane ring. The objective of this study is to develop a bioprocess to produce polyol oils directly from soybean oil. For products separation, we found that a TLC method using a pre-coated thin layer silica gel plate and a two step development solvent systems could separate the polyol products from substrate soy bean oil. The products and substrates were separated in the following order: substrate triacylglycerols (Rf 0.8), free fatty acids (FA, Rf 0.7), product dihydroxy oil (Rf 0.4), trihydroxy oil (Rf 0.3), monohydroxy FA (Rf 0.1) and dihydroxy FA (Rf 0.05). We also found that HPLC with a C18 reverse phase column and a linear gradient of 100% methanol to 100% 2-propanol over 60 minutes at 1 mL/min flow rate was able to separate product polyol oils and substrate soybean oil. Free FA and Polyol oils (Diacylglycerols (DAG) containing hydroxyl FA) were eluted between 5 min to 15 min. DAG containing two normal FA was eluted between 15 min to 28 min and the substrate soybean oil was eluted between 36 min to 45 min. A total of 400 microbial cultures were screened and we found 25 hits. Polyol oils products were purified through a silica gel column chromatography, fractionated by HPLC and then analyzed by MS. A total of 57 molecular species of DAG containing tri-, di-, mono-hydroxy FA and normal FA were identified by MS. HPLC chromatogram of evaporative light scattering detector was used for semi-quantification of these DAG in the purified polyol oils. The total content of the DAG containing two normal FA was about 25% and the total content of DAG containing hydroxy FA might be about 75%. The yield of bioconversion by culture A01-35 from soybean oil to polyol oil products (DAG containing hydroxyl FA) plus DAG containing normal FA was 31% by weight. Bioconversion of soybean oil to polyol oils is a new research area without available methodology to follow. Here for the first time we report the establishment of a new bioprocess for the production of soypolyol oils directly from soybean oil.