Cold acclimation is an adaptive process for acquiring cold/freezing tolerance in wheat. To clarify the cultivar difference of freezing tolerance, we compared mitochondrial respiration activity and the expression profile of alternative oxidase (AOX) genes under low-temperature conditions using two common wheat cultivars differing in freezing tolerance. During cold acclimation, the respiration capacity of the alternative pathway significantly increased in a freezing-tolerant cultivar compared with a freezing-sensitive cultivar. More abundant accumulation of the AOX and uncoupling protein gene transcripts was also observed under the low-temperature conditions in the tolerant cultivar than in the sensitive cultivar. These results suggest that the mitochondrial alternative pathway might be partly associated with the cold acclimation and freezing tolerance in wheat.