The rice stem borer, Chilo suppressalis Walker, which is a key rice pest in northern parts of Iran, overwinters in rice stubble and weeds as mature larvae. Diapause of this pest is initiated between October to November and terminates in March. Seasonal variations in the supercooling point, survival at low temperatures, and sugar contents were studied in field-collected larvae during different phases of diapause. Ambient temperature was lowest in January and February when larvae were at the highest diapause intensity and achieved a high degree of cold hardiness at -10, -15, and -20°C. Glycerol, a major cryoprotectant, reached a peak in January. It appeared that cold hardiness in the larvae is closely associated with the diapause. For the first time, this study suggests that glucose and glycogen are converted to glycerol during cold seasons, but trehalose has no definite role in the interconversion. During the coldest months, supercooling points (SCPs) increased (around — 11°C), and larvae could survive below their SCP values, showing that overwintering larvae of C. suppressalis are freeze tolerant in Iran. Our findings suggest that cold hardiness and diapause are essential components for this species. The ovewintering larvae have high capacity of cold hardiness and can overcome severe winters. Understanding of cold hardiness and overwintering behavior of this species may help in integrated pest management of the rice stem borer in paddy fields.