A novel qualitative method has been developed for the determination of the various types of hydroxyl groups present in biodiesel production streams. The use of 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane as a phosphitylation reagent for quantitative P-31 NMR analysis of the hydroxyl groups in biodiesel process samples has been fully, developed. This experimental protocol allows for rapid analysis of biodiesel mixtures Of alcohols, fatty acids, glycerol, and mono- and disubstituted glycerides. Characteristic chemical shift ranges were developed with model compounds and used to fully characterize the conversion of triglyceride samples to biodiesel for two commercial production processes.