Wheat plants which are exposed to periods of low temperatures (cold acclimation) exhibit increased survival rates when they are subsequently exposed to freezing temperatures. This process is associated with large-scale changes in the transcriptome which are modulated by a set of tandemly duplicated C-repeat Binding Factor (CBF) transcription factors located at the Frost Resistance-2 (Fr-2) locus. While Arabidopsis has three tandemly duplicated CBF genes, the CBF family in wheat has undergone an expansion and at least 15 CBF genes have been identified, 11 of which are present at the Fr-2 loci on homeologous group 5 chromosomes. We report here the discovery of three large deletions which eliminate 6, 9, and all 11 CBF genes from the Fr-B2 locus in tetraploid and hexaploid wheat. In wild emmer wheat, the Fr-B2 deletions were found only among the accessions from the southern sub-populations. Among cultivated wheats, the Fr-B2 deletions were more common among varieties with a spring growth habit than among those with a winter growth habit. Replicated freezing tolerance experiments showed that both the deletion of nine CBF genes in tetraploid wheat and the complete Fr-B2 deletion in hexaploid wheat were associated with significant reductions in survival after exposure to freezing temperatures. Our results suggest that selection for the wild-type Fr-B2 allele may be beneficial for breeders selecting for varieties with improved frost tolerance. © 2013 Springer-Verlag Berlin Heidelberg.