Adequate winterhardiness is crucial for yield stability of asparagus (Asparagus officinalis) cultivars in southern Ontario, Canada, and could be influenced by pattern of the fall fern senescence. Fern of cultivar Guelph Millennium (GM) turns yellow or senesces by mid-October, before that of cultivar Jersey Giant (JG), which often remains green until a killing frost. Early fern senescence could be a signal for cold acclimation competency and consequently winterhardiness, explaining the superior stand longevity and yield observed for GM compared with JG. A field experiment was conducted from mid-August to November to measure physiological parameters related to cold acclimation in fern, rhizome, and storage roots. During fall, fern chlorophyll concentration, rhizome nitrogen concentration, percent water of the crown, and storage root LT50 (temperature at which 50% cell death occurs) decreased. Cultivars did not differ for storage root percent water; however, values were smaller (greater dehydration) for GM than JG in the rhizome. At the end of the sampling period, GM had higher and lower concentrations of rhizome low-molecular-weight, non-structural carbohydrates and sucrose, respectively, than JG, which could support a hypothesis of greater winterhardiness in GM. Storage root LT50 values of –19 °C and the lack of cultivar differences for this trait, in conjunction with differences between GM and JG for rhizome traits thought to be important for freezing tolerance, suggest characteristics of the rhizome in conjunction with timing of fern senescence may be important in cold acclimation of asparagus.