The ability to withstand significant abiotic stresses, such as prolonged freezing temperatures, is vital for the survival of winter wheat (Triticum aestivum L.). Termed ‘winter hardiness,’ this ability develops slowly over the fall growing season as the plant acclimates to the decreasing temperature and day length. Numerous gene families regulate plant response to these environmental cues. Previous studies have identified copy number variation at the VERNALIZATION 1 (Vrn-A1) locus and allelic variation at the FROST RESISTANCE 2 (Fr-A2) locus as significant regulators of cold acclimation. However, significant phenotypic variation for cold hardiness is still observed in winter wheat cultivars with the same VRN-A1/Fr-A2 genotype, suggesting that additional loci could contribute to superior cold hardiness. The objective of this experiment was to examine variability for important cold acclimation traits, including low-temperature tolerance (LT50), vegetative to reproductive transition (VRT), and threshold initiation temperature (IT), using twenty-five representative Canadian winter wheat cultivars with varying combinations of theVrn-A1 and Fr-A2 alleles, copy numbers and haplotypes. LT50 and VRT were determined for each cultivar at ten different acclimation periods (ranging from 7 to 98 days). Artificial freeze tests were utilized to estimate LT50following each acclimation period at 4oC, and IT following a one week acclimation period at six different temperatures (ranging from 4oC to 18oC). VRT was determined by counting final leaf number. These traits provide insights into the acclimation strategies of winter wheat cultivars with varying Vrn-A1/Fr-A2 genotypes, and will assist in the identification of additional loci regulating cold acclimation maintenance in future RNAseq experiments.