The twospotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) is an important agricultural pest. Population dynamics and pest outbreaks highly depend on the overwintering success of the mite specimens; therefore, it is necessary to assess winter survival dynamics of this pest. Seasonal changes in supercooling point (SCP) and acute cold tolerance (2-h exposure at -5, -10, -15, -20, -23, or -25°C) were assessed in field-collected females during the winter in 2010-2011 in Iran. The SCP values varied from a minimum of -30.5°C (January 2011) to a maximum of -12.6°C (April 2011). Significant differences were recorded in the SCP distribution patterns between autumn- and winter-sampled females, depicting the acquisition of cold hardiness over the winter. The mean ambient air temperature was the lowest in January (4°C), when the females showed the highest supercooling ability. Correlated patterns between monthly temperatures and acute cold tolerance also were found. At -20°C, the survival of the mites was very low (10%) when they were sampled in October 2010; whereas it was high (97.5%) in January 2011, before decreasing to 5% in April 2011. The present data show that T. urticae females are chill tolerant and capable of adjusting their cold tolerance over the winter season. Acute cold tolerance (-15 and -20°C) and SCP represent valuable metrics that can be used for predicting the seasonal changes of the cold hardiness of T. urticae females.