Cold acclimation and overwintering state can affect fatty acid compositions of insects. To determine compositional change of fatty acids during nondiapause and diapause stages, an experiment was conducted to investigate fatty acid constituents from whole body of C. suppressalis larvae. Five most abundant fatty acids were found to be palmitoleic (35–58%), palmitic (18–44%), oleic (14–23%), stearic (0.5–2.5%) and linoleic acid (0.4–2%). However, linolenic, erucic, lauric and myristic acid were found at lower level. Saturated fatty acids significantly decreased and conversely unsaturated fatty acids increased from August (pre-diapause) to October (initiation of diapause). The increase in seasonal cold hardiness during cold acclimation, exposed at −15°C for 24 h, was related to degree of fatty acid unsaturation. The elevation of palmitoleic acid content at low temperature resulted in an increase in the overall degree of unsaturation in the whole body. These results indicated the importance of unsaturated fatty acids composition to prepare larvae entering diapause phase.