Perennial ryegrass (Lolium perenne L.) is a coolseason turfgrass species that can exhibit significant freezing injury in northern climatic regions. The objective of this study was to quantify major physiological and biochemical changes occurring during the cold acclimation period in four perennial ryegrass accessions varying in freezing tolerance (two freezing-tolerant accessions, TOL-1 and TOL-2, and two freezing-susceptible accessions, SUS-1 and SUS-2). Plants were subjected to a cold acclimation period of 2°C for 21 d in a controlled environment chamber. Crown tissues were harvested at 0, 7, 14, and 21 d of acclimation for determination of proline, water soluble carbohydrates (WSC), and lipid composition. Leaf photochemical effi ciency (Fv/Fm) was also measured weekly before plant sampling for biochemical components. In general, proline levels increased and Fv/Fm decreased during cold acclimation; however, there were no differences in proline accumulation or Fv/ Fm among the accessions in response to cold acclimation. Water soluble carbohydrates signifi cantly increased in response to 2°C, with TOL accessions (TOL-1 and TOL-2) accumulating higher WSC than SUS (SUS-1 and SUS-2) by 21 d of acclimation. There were also significant changes in major phospholipids classes at 2°C. Specifi cally, TOL-2 exhibited a higher ratio of membrane stabilizing lipids and unsaturated fatty acid content compared to SUS-1, SUS-2, and TOL-1 by 21 d of cold acclimation.