Winter survival is an essential trait for winter wheat (Triticum aestivum L.) cultivars grown in high latitude regions such as Eastern Canada. Indoor studies have identified that copy number variation of genes influencing freezing is an essential component. Although Canadian winter wheat is predominantly grown in Eastern Canada, the extent to which allele variation in freezing tolerance genes affects winter survival in this region remains unknown, as there are presently no studies characterizing such variation in Canadian winter wheat germplasm. In this study, we characterized a panel 415 Canadian winter wheat cultivars for haplotype diversity of the Frost Resistance-2 (Fr-A2) locus and copy number variation of Vernalization-A1 (Vrn-A1) and C-repeat binding factors-A14 (CBF-A14). Additionally, this study evaluates each gene’s effect on winter survival across two locations and 2 years. We found that a combination of Vrn-A1 copy number and Fr-A2 haplotype accounted for 67.38% of the genotypic variance. Most of the cultivars tested (77.3%) carry the allele combination of three copies of Vrn-A1 and the Fr-A2-T haplotype, which was associated with the best winter survival. Interestingly, copy number of Vrn-A1 did not significantly affect heading time, therefore, selecting for higher copy number of Vrn-A1 would not affect maturity.