Landry, E. J. and Wolyn, D. J. 2012. A method to assess cold acclimation and freezing tolerance in asparagus seedlings. Can. J. Plant Sci. 92: 271-277. Assessment of winter-hardiness using field-grown asparagus is complicated by variable, yearly climatic conditions and the large crown growing below the soil surface. The development of a seedling assay in controlled environments would be beneficial to study the physiology of winter-hardiness and to facilitate the selection of superior genotypes in breeding programs. Two cultivars, Guelph Millennium (GM) and Jersey Giant (JG), with differing patterns of autumn fern senescence in the field, where GM senesces earlier than JG, were compared. Seedlings were analyzed for physiological parameters after cold acclimation (10°C day/5°C night) or cold acclimation followed by sub-freezing (3°C to -3°C) in controlled environment chambers. Cold acclimation induced greater chlorophyll loss in GM than JG, consistent with previous field observations. LT₅₀, the temperature at which 50% mortality occurs, decreased to approximately -8°C for both cultivars after the initial cold acclimation treatment. Subsequent subfreezing acclimation increased the LT₅₀ for JG to -5°C, decreased freezing tolerance, while that for GM did not change. Early senescence and high proline concentration as well as stable protein and reducing sugar concentrations were associated with the freezing tolerance observed in GM. Further studies are required to establish if the cultivar differences for freezing tolerance identified here are correlated with experiments from field-grown plants.