Adequate winter hardiness is critical for persistence and yield of ' Asparagus officinalis' L. cultivars in Southern Ontario, Canada, and may be influenced by fall senescence. Cultivar 'Guelph Millennium' (GM) turns yellow by mid-October prior to 'Jersey Giant' (JG), which will often 'freeze green'. Field and growth chamber experiments were conducted to understand the relationship between fall senescence and freezing tolerance. Shortened photoperiod and night temperatures below 10°C resulted in a significant increase in freezing tolerance and metabolites, and decreased percent water and chlorophyll concentration. Chlorophyll concentration declined and crown protein and proline concentrations increased earlier for field acclimated GM than for JG. The two cultivars did not have significantly different LT 50 values when field acclimated. However, after sub-freezing acclimation GM seedlings showed significantly higher freezing tolerance than JG. Tolerance to these imposed freeze-thaw events and timely senescence could enhance winter hardiness of asparagus.