The adaptation of St. Augustinegrass (Stenotaphrum secundatum [Walt.] Kuntze) in the transitional climatic zone of the United States is limited due to a lack of sufficient freeze tolerance. Lab-based freeze testing provides plant breeders with a reliable method for evaluating freeze tolerance. However, in developing protocols for freeze testing it is important to account for the underlying mechanisms that affect freeze tolerance. The objective of this research was to evaluate the effects of cold acclimation and deacclimation on nine St. Augustinegrass genotypes ranging in their levels of freeze tolerance. Results indicated that accounting for all levels of acclimation provided excellent genotype separation across freezing temperatures (− 3 and − 4 °C) and supports the hypothesis that the inclusion of acclimation response offers the best overall assessment of freeze tolerance. Genotypes with the highest known field winter survival had also the highest improved freeze tolerance upon cold-acclimation. Other genotypes did not respond to cold-acclimation which resulted in poor survival and recovery rates. Conversely, a significant loss of freeze tolerance was identified when plants were subjected to deacclimation events suggesting that St. Augustinegrass can be negatively affected by rapid temperature changes in the transitional climatic zone leading to increased sensitivity to winterkill. Overall, this study provides preliminary information regarding the complex relationships within and between mechanisms affecting freeze tolerance in St. Augustinegrass. Moreover, results presented here should aid in the development of protocols for selection of freeze tolerant breeding materials under controlled environmental conditions.