Asparagus (Asparagus officinalis L.) cultivars differ for adaptation in southern Ontario, and delayed or decreased acquisition of freezing tolerance in the fall could explain, in part, the diminished longevity observed in some germplasm. A field study was conducted to determine the relationship between LT₅₀, the lethal temperature at which 50% of plants die, and physiological parameters related to freezing tolerance, in three cultivars with varying adaptation in southern Ontario: Guelph Millennium (GM) > Jersey Giant (JG) > UC 157 (UC). The experiment was replicated at two sites in one location, in each of two years. LT₅₀ values for GM were lower (increased freezing tolerance) than those for UC in early October; levels for JG were intermediate. In late-October and early-November, the cultivars did not differ. Increased freezing tolerance was associated with high low-molecular-weight fructan (LF), protein and proline concentrations and low sucrose concentration in the rhizome, and high sucrose and proline concentrations and low LF concentration in the storage roots. Acclimation traits were generally consistent over years and deviations may have been related to differing temperature profiles. Results indicate that winter survival of asparagus is in part determined by timely fall acclimation allowing plants to survive exposure to early frosts.