Little is known about the capacity of Cannabis sativa to cold acclimate and develop freezing tolerance. This study investigates the cold acclimation capacity of nine C. sativa varieties and the underlying genetic and epigenetic responses. The varieties were divided into three groups based on their contrasting cold acclimation capacities by comparing the survival of non-acclimated and cold-acclimated plants in whole-plant freeze tests. In response to the cold acclimation treatment, all varieties accumulated soluble sugars but only the varieties with superior capacity for cold acclimation could maintain higher levels throughout the treatment. In addition, the varieties that acclimated the most efficiently accumulated higher transcript levels of COR genes and genes involved in de novo DNA methylation while displaying locus- and variety-specific changes in the levels of H3K9ac, H3K27me3 and methylcytosine during cold acclimation. Furthermore, these hardy C. sativa varieties displayed significant increases in methylcytosine levels at COR gene loci when deacclimated, suggesting a role for locus-specific DNA methylation in deacclimation. This study uncovers the molecular mechanisms underlying cold acclimation in Cannabis sativa and reveals higher levels of complexity regarding how genetic, epigenetic, and environmental factors intertwine.