Heat stress due to high daytime temperatures is one of the main limiting factors in rice (Oryza sativa L.) yield in Colombia. Thus, the objective of the present research was to analyze the effect of three different daytime temperatures (25, 35, and 40 °C) on the physiological responses of three Colombian rice cultivars (F60, F733, and F473), thereby contributing to the knowledge of rice acclimation mechanisms. For 10 d, eight plants of each of the three cultivars were subjected daily to 5 h periods of 35 and 40 °C. The control treatment corresponded to normal growth conditions (25 °C). Thermal stress was assessed based on a series of physiological and biochemical parameters. The 35 °C treatment produced photosynthetic and respiratory differences in all three cultivars. At 40 °C, ’F60’ displayed the lowest photosynthetic rate and the highest respiratory rate. Although this cultivar experienced particularly strong electrolyte leakage and changes in proline when subjected to the high-temperature treatments, similar trends were observed in ’F733’ and ’F473’. At 40 °C, the concentration of malondialdehyde (MDA) was lower in ’F473’ than in the other cultivars. These results may explain the poor agronomic performance of ’F60’ in the field under daytime heat stress. The methodologies employed in the present work may be useful in Colombian rice breeding programs, particularly for the selection of heat-tolerant breeding stocks.