In this study we investigated the heat tolerance of high yielding Australian cotton (Gossypium hirsutum L.) cultivars using a multi-level approach encompassing physiological assays and measurements of performance. Two cultivars with known field performance were evaluated for heat tolerance under optimal (32°C) and high (42°C) temperatures in a growth cabinet with a cell membrane integrity assay. Impacts of temperature on growth were evaluated with leaf level measurements of gas exchange and chlorophyll fluorescence. To extend the multi-level approach, the expression of a Rubisco activase regulating gene (GhRCAα2) was also determined. Consistent with previously determined differences in the field, cultivar Sicot 53 outperformed Sicala 45 for the cell membrane integrity assay; this finding was reflective of cultivar differences in gas exchange and chlorophyll fluorescence. Cultivar differences were also consistent for expression of GhRCAα2, which may also help explain differences in physiological performance, particularly photosynthesis. This study reaffirmed that physiological and molecular assays were sufficiently sensitive to resolve genotypic differences in heat tolerance and that these differences translate to physiological performance. By comparing performance under high temperatures in the growth cabinet and field, this approach validates the use of rapid screening tools in conjunction with a multi-level approach for heat tolerance detection.