Understanding cold acclimation and identifying the low molecular weight carbohydrates that support the development of freezing tolerant safflower seedlings will aid in breeding winter-hardy cultivars for temperate cropping systems. Three field selected lines of winter safflower (WSRC01: PI 651878; WSRC02: PI 651879; WSRC03: PI 651880) were cold acclimated for four weeks at 4 °C and compared to seedlings grown for two weeks at 20 °C. The commercial spring-type cultivar, Olé, served as a non-hardy check. Leaf, stem, and root fructose, glucose, sucrose, raffinose, and stachyose concentrations all increased to variable extents across the PI accessions after cold acclimation. In comparison with Olé, winter safflower accessions tended to be more responsive to cold acclimation by increasing metabolite concentration. Verbascose was only recovered within leaf tissue and PI 651880 was the only entry to show a substantial alteration in verbascose concentration due to cold acclimation. Based on these data, no specific low molecular carbohydrate was responsive or responsible for the accumulation of freezing tolerance, but a concert of metabolites and their responsiveness may help explain the observed differences in development, freezing tolerance, and ultimately winterhardiness among safflower germplasm.