Plants are constantly subjected to adverse environmental conditions that alter their growth and productivity, with an estimation that approximately 50% of annual average crop yields are reduced due to abiotic stresses. Freezing stress causes desiccation and ice damage in plants and is becoming more important as temperatures and unpredictable weather patterns increase. Normally, plants acclimate to cold temperatures as winter approaches and deacclimate as temperatures warm in the spring. Cold acclimation in fall is required for plants to build up their cellular defenses against desiccation and intracellular ice formation, while deacclimation is the process in which plants metabolize protective compounds for regrowth during spring. Recent trends in weather conditions, including increased frequency of winter temperature extremes, suggest an increase in issues associated with lower cold acclimation capacity and higher rates of premature deacclimation. Although cool-season temperate crops are usually tolerant to low temperatures, some species are sensitive to winter temperature extremes. Therefore, research into mechanisms associated with overwintering is crucial to better understand how to maximize plant overwintering survival. Freezing tolerance has been difficult to characterize due to its complexity, myriad stresses associated with it, and the balance between cold acclimation and deacclimation. The objectives of this dissertation research were to study the different physiological and molecular mechanisms associated with cold acclimation and deacclimation resistance in perennial ryegrass (Lolium perenne) differing in freezing tolerance capacities. We screened plants to investigate the genetic variability in freezing tolerance and deacclimation resistance, which resulted in us identifying two genotypes that had the greatest differences in their responses to cold acclimation and deacclimation. We further found that the genotype sensitive to deacclimation had a higher crown moisture content and photosynthetic activity during cold acclimation and deacclimation, which could predispose the genotype to rehydration and regrowth during warming events. Lastly, we examined global transcriptome responses to deacclimation, and identified biological processes and pathways that could account for differences seen in deacclimation resistance, which include growth, photosynthesis, and flower development. These results provide information on physiological and transcriptomic responses for overwintering in perennial ryegrass, which can improve management practices and breeding programs for cultivar selection.