Winterhardiness is an important trait for asparagus (Asparagus officinalis) cultivars grown in temperate climates. Several biochemical and physiological parameters are correlated with the acquisition of freezing tolerance during cold acclimation in the fall and could be used as indirect measures for selection in a breeding program. Genetic variation was assessed before and after fall acclimation in August and November, respectively, for freezing tolerance attributes in 18 asparagus hybrids and 24 clones, which included male, supermale, and female genotypes. Fern chlorophyll and rhizome sucrose concentrations and storage root and rhizome percentage water decreased, whereas the concentrations of storage root proline, glucose and sucrose, and rhizome proline and high-molecular-weight fructan increased during the fall. Germplasm did not differ in August but significant variation was observed in November for most parameters, indicating genotype-specific responses to fall acclimation and the acquisition of traits associated with freezing tolerance. Narrow-sense heritability estimates were significant for fern chlorophyll, storage root proline, and rhizome glucose and sucrose concentrations. With significant genetic variation and heritability, breeding to improve freezing tolerance could be possible with indirect selection measures.