Understanding cold acclimation (CA) is important for concurrently improving autumn yield and winter survival in alfalfa (Medicago sativa L.). Medicago truncatula Gaertn., an annual relative of alfalfa, could be used to determine genetic bases of CA, if the ability and conditions required for its CA are determined. The major objective of this study was to develop a laboratory screening procedure to quantify CA in M. truncatula. Two genotypes, Jemalong-6 and W6 5018, were grown in nonacclimation (NA) and three CA regimes (CA1, CA2, and CA3). CA was quantified by measuring freezing tolerance [LT50 (the freeze temperature at which 50% injury occurred)], as estimated by ion leakage (IL) from leaf tissues. The percentage of injury and LT50 were derived from freeze injury data. Cold-acclimated plants had reduced stem length, number of leaves, stem dry weight, leaf dry weight, and root dry weight compared with control. Root-to-shoot ratio was higher in cold-acclimated than in control plants. These results indicate the clear initiation of acclimation response in cold-acclimated plants. Average LT50 temperatures were −3, −8, −7.8, and ≥ −12.5 °C in NA, CA1, CA2, and CA3 regimes, respectively. While 80% injury was induced by −7 °C in NA plants, −20 °C only induced an average of 52% injury in CA3 plants. While the percentage of injury was lower in Jemalong-6 than in W6 5018 for all CA regimes, CA3 regime was most effective in distinguishing CA ability of the two genotypes. Our results demonstrated the capability of M. truncatula to cold acclimate under the controlled CA regimes and the possible use of IL as a rapid laboratory method to quantify CA.