Display omitted •GC–MS sugar/polyol pattern for non- vs. diapausing O. nubilalis larvae was analyzed.•Glycerol, trehalose, sorbitol, mio-inositol, glucose and fructose were identified.•Glycerol and trehalose are the most abundant compounds in diapausing larvae.•In mid diapause, cold induced elevated glycerol and reduction of other polyols.•Low temperature increased content of all tested sugars at the end of diapause. The European corn borer (ECB, Ostrinia nubilalis Hbn.) is a major pest in temperate regions of Europe and North America. Fifth instar ECB larvae enter diapause before winter and gradually develop cold hardiness. Here we investigated the combined influence of diapause phase and low temperature on sugar and polyol content in ECB larvae. Larvae in mid-diapause or diapause termination were acclimated at 5 °C, −3°C or −16 °C, and sugar and polyol content was measured using GC–MS. Control GC–MS measurements were conducted on untreated non-diapausing larvae. We detected differences in polyol (glycerol, sorbitol, myo-inositol) and sugar (trehalose, fructose, glucose) levels in diapausing versus non-diapausing larvae. Glycerol and trehalose were the most abundant of all analyzed cryoprotective compounds in diapausing larvae. Exposure of diapausing larvae to decreasing temperatures induced changes in polyol and sugar levels that depended on the phase of diapause. In mid-diapause larvae, decreasing temperatures induced a significant increase in glycerol and a decrease in sorbitol and myo-inositol. In larvae at diapause termination, polyol content was lower and less influenced by decreasing temperatures. In contrast, sugar levels were lower in larvae at mid-diapause versus diapause termination. Exposure of larvae to −16 °C induced a significant increase in the levels of all detected sugars. In particular, glucose levels were significantly higher in larvae at diapause termination following exposure to −16 °C. We propose that this shift toward sugar synthesis following low temperature exposure in larvae at diapause termination is a consequence of NADPH dependent polyol synthesis, and may be a mechanism for preservation of carbon reserves needed for post-diapause development.