The gene family of C-repeat binding factors (CBFs) has been established to play a prominent role in cold acclimation in plants. The expression of CBF genes in hexaploid wheat was investigated by comparing the response to cold acclimation of four near-isogenic lines previously generated from two cultivars, spring Manitou (a non-hardy spring wheat) and winter Norstar (a very cold-hardy winter wheat), wherein the major vernalization locus Vrn-A1 was swapped. The CBF cluster on the linked Fr2 locus was not included in the swapped Vrn-A1 region, thus was fixed in the Norstar and Manitou backgrounds. These lines allowed us to examine the effect of the Vrn1 locus on the expression of 16 CBF genes without the confounding effects of genetic variations outside of the swapped region. We showed that CBFs exhibit different temporal regulations during cold treatment, hence were not functionally equivalent. The expression of four of the CBFs was not detected in the crown. CBFI, CBFII-5.2, CBFIIIc-B10 and CBFIII-D3 expressions were down-regulated in all four genotypes. Members of CBFIIId and CBFIVb-d subgroups were up-regulated with expression profiles that showed variable dynamics supporting different transcriptional and/or post-transcriptional controls. Analysis of the CBF expression profiles during cold-acclimation generated hypotheses as to which of the CBF genes are more likely to be involved in the development of low temperature tolerance in cereals. We also provide further evidence that the Vrn-A1 locus acts as a negative regulator of CBF genes.