Kim, J. and Wolyn, D. J. 2015. Freezing tolerance assessment for seedlings of three asparagus cultivars grown under controlled conditions. Can. J. Plant Sci. 95: 495-504. Asparagus (Asparagus officinalis L.) cultivars grown in southern Ontario must be winter-hardy. Development of a method to screen seedlings for freezing tolerance directly, or indirectly through metabolite analysis, could be useful in a breeding program. Ten-week-old seedlings of three cultivars with varying adaptation to southern Ontario, Guelph Millennium (GM), Jersey Giant (JG) and UC157 (UC), were acclimated under factorial combinations of two temperatures (7 or 23°C) and two photoperiods (8 and 16 h), with or without 5 additional days of sub-freezing acclimation at 3/-3°C (12/12 h) in darkness. Plants were then evaluated for metabolites and LT₅₀, the temperature at which 50% of plants die. Photoperiod had no effect, but low temperature without sub-freezing acclimation decreased LT₅₀ (increased freezing tolerance) of all three cultivars. The ranking of freezing tolerance, GM>JG>UC, was consistent with observed persistence in the field. The cultivars differed for concentrations of fern chlorophyll, and crown proline, high-molecular-weight fructan and sucrose, as well as crown percentage water. LT₅₀ was highly correlated with crown percentage water, and chlorophyll, proline, sucrose, and high-molecular-weight fructan concentrations, suggesting these traits could be used as indirect measures to breed for winter-hardy cultivars.