Description: In the literature, frost hardiness (FH) studies in trees have often been restricted to one organ (buds, leaves, needles or twigs). To extend our knowledge and gain a unified view, FH differences between organs and tissues or throughout the life of the tree have to be characterized in relation to physiological changes. In this study, different organs and tissues of young potted and mature orchard walnut trees ( Juglans regia L.) were compared for seasonal changes in FH during different years. FH was assessed using the electrolyte leakage method. Physiological parameters were concomitantly monitored focusing on two significant traits: water content (WC) and carbohydrate content (glucose + fructose + sucrose, GFS). No seasonal variation in FH was observed in the root system, but acclimation and deacclimation were observed aboveground. Among organs and tissues, cold sensitivity levels were different in deep winter, with buds most sensitive and bark most resistant, but acclimation/deacclimation dynamics followed similar patterns. Physiological variation was also similar among organs: FH increased when WC decreased and/or soluble carbohydrates increased. Based on these results, relations between soluble carbohydrate content, WC and FH were calculated independently or in interaction. The key results were that: (i) the relationship between FH and physiological parameters (GFS and WC), which had previously been shown for branches only, could be generalized to all aboveground organs; (ii) lower WC increased the cryoprotective effect of GFS, showing a synergic effect of the two factors; (iii) the best fit was a non-linear function of WC and GFS, yielding a predictive model with an root mean square error of 5.07 °C on an independent dataset and 2.59 °C for the most sensitive stages; and (iv) the same parameters used for all organs yielded a unified model of FH depending on physiology, although the variability of GFS or WC was wide. The model should be of value for predicting FH in walnut independently of previous growing conditions (i.e., after sublethal stress accumulation).