The pistachio seed wasp, Eurytoma plotnikovi Nikol'skaya (Hymenoptera: Eurytomidae), is one of the main pests in various pistachio growing regions of Iran. This pest passes the winter as diapausing last instar larvae. In this study, the relationship between diapause and cold hardiness and also the physiological and biochemical characteristics the diapausing and nondiapausing larvae of E. plotnikovi were investigated. Digestive α-amylase enzyme showed a high activity (70.41 ± 2.36 μg maltose/min per mg protein) in nondiapausing larvae, but its activity vigorously decreased during the diapause period. Glycogen declined at the beginning of diapause until March. Decrease in glycogen content was proportional to increase in total simple body sugars, trehalose, myo-inositol, and sorbitol contents. Lipid accumulated from the onset of diapause in September until January reaching a high concentration of 28.74 mg/g fresh body weight, but then declined from March to end of diapause in April.The supercooling points were decreased from August (–17.68 ± 0.14°C) to January and reached to its lowest point in January (–23.14 ± 0.27°C), the coldest month of the year, then gradually increased through April (–21.38 ± 0.32°C). The survival rates at low temperature indicate that last instar larvae of E. plotnikovi are most cold tolerant in December–February when total body sugars, trehalose, myo-inositol, and sorbitol concentration is high, suggesting an alternative cryoprotective role for these compounds. The experimental data show that E. plotnikovi is freeze avoidance insect.