Temperature of a plant organ constitutes an integrative index to its eco-physiological properties and status. However, little attempt has been made to dissect the combined effect of ecological and physiological factors on the surface temperature of a plant organ such as the rice spikelet. In this study, using a deactivated plant as reference, we developed a novel comparison method to dissect the environmental and physiological effects on temperature of rice spikelet. Three japonica rice cultivars with contrasting canopy features were used as testing materials. Temperatures of flag leaf, superior and inferior spikelets and their diurnal rhythm during grain filling stage were precisely measured by a hand-held infrared thermometer. The results showed that the variation of environmental conditions within a panicle was relatively minor, posing a limited influence on temperature difference between the superior and inferior spikelet. On the other hand, it was the intrinsic physiological properties that considerably affected the spatial variations of spikelet temperature within a panicle. Chemical analysis of sucrose and starch in grains and bracts indicated that the superior spikelet is more physiologically active at photosynthetic assimilation and starch biosynthesis. Interestingly, sugar in bracts exhibited a pattern of diurnal changes similar to the source leaf but different from the sink grain, confirming that bracts are source organs for grain filling. Our findings yield penetrating insight into the eco-physiological foundation of spikelet temperature, thus being helpful for the application of physiological approaches in crop breeding for cooler canopy.