The almond wasp, Eurytoma amygdali Enderlein (Hymenoptera: Eurytomidae), a key pest of almond, is a univoltine pest diapausing as last instar larvae inside the damaged fruits for almost nine months in a year. In this study, changes in the amount of total simple sugars, lipid, protein, glycogen, trehalose, glucose, supercooling points (SCPs), and cold hardiness of the diapausing larvae were measured from October to March for first year diapause-destined and in August and September for second year diapause-destined larvae. Changes in glycogen content were reversely proportional to changes in total simple sugars and low molecular weight carbohydrates. These changes reflect the interconversion of glycogen to sugar alcohol in order to increase the insect cold tolerance. We found that cold hardiness and diapause of the last instar larvae of the almond wasp have evolved separately. Cold hardiness was highly associated with physiological changes (accumulation of cryoprotectants), but no physiological changes occurred in early diapause of first year diapause-destined and second year diapause-destined larvae. The almond wasp larvae were found to be a freeze-avoidant insect, as no larva survived after SCP determination and crystallization of its body fluids.