Raman spectroscopy is used for studying the composition of cell lipidome. However, the Raman approach suffers from limited distinguish power for different lipids. In recent years, isotopically labeled molecules are increasingly used to enhance the Raman resolution of similar compounds in biological samples. Here, we present Raman study of fully deuterated stearic acid (stearic‐d35), semi‐deuterated oleic acid (oleic d‐9), arachidonic acid with deuterated methine groups (arachidonic‐d8), and their hydrogenated analogs. Polarization properties and temperature dependences of CD stretching modes were investigated to reveal the capability of the isotopically shifted Raman peaks in molecular characterization. Sensitivity of Raman peaks to order–disorder transition corresponding was demonstrated for deuterated methine, methylene, and methyl groups. It was found that the isotopically shifted Raman peaks provide information about the orientation of deuterated Raman tags and their surroundings. Highlights: The assignments for Raman peaks at 1150 and 992 cm−1 of deuterated hydrocarbons are verified.Polarized Raman spectra for crystalline deuterated stearic acid are investigated.Polarization dependence of ═CD, CD2, and CD3 Raman peaks is studied.Temperature dependences for deuterated ═CD, CD2, and CD3 Raman peaks were measured.Sensitivity of ═CD, CD2, and CD3 modes to the phase transitions is analyzed. [ABSTRACT FROM AUTHOR]