Heat stress is the major abiotic stressor in agriculture which reduces crop productivity and yield. Six sugarcane (Sacharum officinarum L.) genotypes were studied to investigate the impact of three temperature levels at four phenological stages on tissue electrolyte production and the feasibility of using the cell thermostability method (CTM) for the identification and selection of heat tolerant sugarcane genotypes. The cell membrane thermostability was quantified by measuring relative cell injury percentage with a modification in the temperature treatment on four phenological stages in a field experiment. Our results suggest that heat tolerance based on cell membrane thermostability can be improved using the existing genetic variability available within the commercial or experimental sugarcane germplasm. We conclude that the cell membrane thermostability test can be a useful screening procedure for selecting sugarcane genotypes that tolerate high temperature stress. The test can be used in conjunction with a temperature trait of 60 °C during the maturity stage. This procedure predicts the ability of sugarcane genotypes to maintain yield and juice quality under stressful field conditions.