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To investigate the influence of winter climate on freezing tolerance at the population level, minimum January air temperatures in the field and cold acclimation determined in the laboratory were compared for Opuntia fragilis. Populations occurred at 20 locations as far north as 56-degrees-46’ N latitude and at elevations up to 3029 m in Canada and the United States, most of which experience extreme freezing temperatures each winter. Low-temperature responses and water relations of stems were examined in the laboratory at day/night air temperatures of 25-degrees/15-degrees-C and 14 d after the plants were shifted to a 5-degrees/-5-degrees-C temperature cycle. Cold acclimation averaged 17-degrees-C and freezing tolerance averaged -29-degrees-C for the 20 populations following a shift to low day/night air temperatures, indicating that 0. fragilis has the greatest cold acclimation ability and the greatest freezing tolerance reported for any cactus. Moreover, freezing tolerance and cold acclimation were both positively correlated (r2 congruent-to 0.7) with the minimum temperatures at the 20 locations. Plants lost water during low-temperature acclimation, leading to 30% decreases in cladode and chlorenchyma thickness; the decrease in water content was greater for the five warmest populations than for the five coldest ones. Over the same period, the average osmotic pressure of the chlorenchyma increased from 1.42 to 1.64 MPa, and the relative water content (RWC) decreased from 0.58 to 0.49, but the average osmotic pressure of saturated chlorenchyma was unchanged, indicating no net change in solute content during acclimation. Although the role of water relations in freezing tolerance is unclear, the substantial freezing tolerance and cold acclimation ability of O. fragilis leads to its distribution into regions of Canada and the United States that experience minimum temperatures below -40-degrees-C during the winter.