AbstractImportant factors and new techniques in the application of ozonolysis to the localization of double bond positions in unsaturated fatty acids are elaborated. Solvent polarity was shown to exert a profound influence on the yield and composition of ozonides. The influence of fatty acid structure on ozonide formation and the thin‐layer chromatography (TLC) of ozonides of a number of different types of unsaturated fatty acids are illustrated. Simple methods for the ozonization of unsaturated fatty acid esters and the cleavage of ozonides by reduction and pyrolysis are described. Applications of ozonolysis to the determination of the structures of geometric isomers, hydroxy, epoxy and cyclic olefinic fatty acids, and the localization of radioactivity along the chain of unsaturated fatty acids are illustrated.