Methods used for assessing survival of winter wheat under low temperature flooding and ice encasement (LTFIE) are limited. This study developed unique methods to determine survival and characteristics associated with survival of wheat cultivars under LTFIE. Acclimated cultivars AC Carberry, Branson, CM614, and Norstar were used to estimate the days to reach 50% mortality (LD50) and the temperature to reach 50% mortality (LT50) under ice. Norstar had the longest LD50, Branson and CM614 had similar LD50s, and AC Carberry did not reach an LD50. All four cultivars had different LT50s, Norstar had the lowest and AC Carberry had the highest. Branson, CM614, and Norstar had the highest sucrose, simple sugar, fructan, and total water-soluble carbohydrate (TWSC) concentrations when acclimated and encased in ice. Reliability of methods were confirmed based on the cultivars’ LD50, LT50, and WSC concentrations. This research will aid in testing for LTFIE using the developed methods.