Cold stress is a major abiotic factor plants face during their life cycle. Although plants often exhibit phenotypic variation in cold tolerance, the underlying mechanism remains poorly understood. In the present study, the 50% lethal temperature (LT50) values of 37 Arabidopsis thaliana accessions at latitudes from 15° to 58° ranged from -13.2°C to -4.9°C and were closely correlated with the cold climates of the collection sites. According to a methylation analysis of all C-repeat (CRT)-binding factor (CBF) pathway genes, the coding and promoter regions of AtICE1, a regulator of CBF genes, exhibited the greatest variability in methylation levels among the accessions and included 5-122 methylated cytosine residues. In contrast, unmethylated or only slightly methylated genes in the CBF pathway showed little variation among the accessions. According to a gene expression analysis of four selected A. thaliana populations with distinct methylation patterns, except for the down-regulated gene AtCBF2, the expression levels of all members of the CBF pathway were negatively correlated with AtICE1 gene methylation levels. Treatment of the four A. thaliana populations with the DNA methylation inhibitory reagent 5-azacytidine resulted in a 30.0-78.3% enhancement of freezing tolerance and decreases in LT50 values of approximately 1.9-3.6°C. Similar effects were observed in drm2 mutants, including 30.0-48.3% increases in freezing tolerance and decreases in LT50 values of approximately 0.7-3.4°C. Thus, the AtICE1 methylation-regulated transcription of CBF pathway genes is responsible for the phenotypic variation in the freezing tolerance observed in A. thaliana.