Through the annual cycle of plant growth and dormancy, the winter season leads to profound metabolic changes allowing plants to undergo cold acclimation. In boreal environments, winter conditions are changing rapidly and are likely to cause damage to commercial wild lowbush blueberry. In this study, we addressed the level of frost hardiness and determined the role of environmental factors and nonstructural carbohydrates (NSCs) on frost hardiness. From autumn to spring, stem sections of Vaccinium angustifolium and Vaccinium myrtilloides were harvested each month in a commercial blueberry field to assess the relative electrolyte leakage and calculate the temperature at which 50% of the cells are lysed [LT50 (degrees C)], used as frost hardiness index. Stems were also collected to assess soluble carbohydrates and starch. Correlations, principal component analysis (PCA) and structural equation modeling (SEM) were used to determine how environmental factors and NSCs directly or indirectly influence the frost hardiness index. Frost hardiness reached its lowest level in December and January with LT50 dropping below -60 degrees C. Seasonality of frost hardening was closely linked to photoperiod and temperature, generating clock-wise hysteretic loops that divide frost hardening into acclimation, from September to January, and deacclimation, from January to the end of May. Environmental factors such as photoperiod and temperature were more important in determining the level of frost hardiness during acclimation, with either direct or indirect effect through an influence on starch degradation, increasing soluble carbohydrate content. During deacclimation, soluble carbohydrates, especially raffinose, further induced a stronger direct regulation of frost hardiness. Direct biological regulation through raffinose defined the level of frost hardiness during deacclimation. However, the negative influence of temperature on raffinose concentration could increase vulnerability to winter warming events.