The C-Repeat Binding Factors (CBFs) are transcriptional activator proteins that play a key role in effecting freezing tolerance in plants. They were originally identified from Arabidopsis thaliana using a functional screen for proteins capable of binding to the CRT/DRE, a cis-acting low temperature DNA regulatory element. Approximately 4% of the Arabidopsis protein-encoding genes are directly or indirectly regulated by the CBFs. Activation of this set of genes leads to increased freezing tolerance. Addressing what is the role of the CBFs in effecting freezing tolerance in barley and wheat is revealing a complex story in which differences in CBF gene copy numbers is a central theme. On the long arm of group 5 chromosome homoeologs is a cluster of 11 – and possible more – distinct CBF gene orthologs. Approximately half of these orthologs are duplicated, existing as identical or nearly-identical paralogs in individual genomes. Moreover we find that the genomic region encompassing subsets of these orthologs are amplified in winter genotypes but not in spring genotypes. In winter barleys there are two to eight copies of a 22 kb genomic segment encompassing CBF2A and CBF4B. Spring barleys in contrast harbor single copies of CBF2 and CBF4. In winter wheats there are increased copy numbers of CBF14, while spring wheats harbor single copies – and these distinctions occur on all three homoeologs. To gain insight into the role copy number variation plays in freezing tolerance of these cereals we are using approaches utilizing unstructured mapping populations and Agrobacterium-mediated transformation. The results from these studies are offering mechanistic insight into how we might go about breeding for enhanced freezing tolerance and winter survival. This work was supported by grants from the Ohio Plant Biotechnology Consortium (2010-011) and USDA-CSREES subaward CO396A-F.