Species distributed across diverse climate and thermal conditions represent opportune systems for studying tolerance of low temperature stress. We examined variation in cold acclimation capacity and freezing tolerance among three natural populations (Texas, Kansas, and Manitoba) of the perennial sunflower species Helianthus maximiliani, originally collected across 2134 km in central North America. Tolerance to low temperatures was evaluated through leaf electrolyte leakage assays that quantify loss of cellular electrolytes into an aqueous medium due to plasma membrane damage. Freezing tolerance was highest for plants from the northernmost latitude (Manitoba population) under both non cold- acclimated and cold- acclimated experimental conditions. Individuals from Kansas and Texas populations exhibited lower freezing tolerance compared to Manitoba but did not differ from one another. Plants from all populations retain the ability to increase freezing tolerance through the process of cold acclimation. Freezing tolerance of Manitoba × Texas F1 hybrids was statistically indistinguishable from plants from the Texas population and possible explanations for these observations are discussed. Analysis of flowering specimens from herbaria records of corresponding regional locations indicates considerable variation in flowering phenology whereby flowering occurs progressively earlier with increasing latitude. This phenological variation may provide an additional mechanism of coping with low temperature stress through temporal avoidance.