As the seasons progress, autumn-planted winter wheat plants (Triticum aestivum L.) first gain then progressively lose freezing tolerance. Exposing the plants to freeze–thaw cycles of -3/3 °C results in increased ability to tolerate subsequent freezing to potentially damaging temperatures. This study was conducted to determine to what extent the length of time that a plant is grown at low temperatures influenced the effectiveness of this freeze–thaw enhancement of freezing tolerance. Plants from six winter wheat lines were grown at 4 °C for 1–18 wk, exposed to 0–2 cycles of freezing to -3 °C for 24 h, then thawed for 24 h at 3 °C, then tested for their ability to tolerate freezing to -10 °C to -17 °C. The freeze–thaw treatments resulted in increased freezing tolerance after 6–12 wk of growth at low temperatures, but had no significant effect before or after that time period. Two cycles of -3/3 °C freeze–thaw was consistently more effective than one cycle. Variation in the extent and timing of the effectiveness of the freeze–thaw treatments was found among the wheat lines, suggesting genetic variation that may be useful for prolonging freezing tolerance further into the winter months could be found in winter wheat.