Abstract Background Our understanding of the physiological responses of rice inflorescence (panicle) to environmental stresses is limited by the challenge of accurately determining panicle photosynthetic parameters and their impact on grain yield. This is primarily due to the lack of a suitable gas exchange methodology for panicles and non-destructive methods to accurately determine panicle surface area. Results To address these challenges, we have developed a custom panicle gas exchange cylinder compatible with the LiCor 6800 Infra-red Gas Analyzer. Accurate surface area measurements were determined using 3D panicle imaging to normalize the panicle-level photosynthetic measurements. We observed differential responses in both panicle and flag leaf for two temperate Japonica rice genotypes (accessions TEJ-1 and TEJ-2) exposed to heat stress during early grain filling. There was a notable divergence in the relative photosynthetic contribution of flag leaf and panicles for the heat-tolerant genotype (TEJ-2) compared to the sensitive genotype (TEJ-1). Conclusion The novelty of this method is the non-destructive and accurate determination of panicle area and photosynthetic parameters, enabling researchers to monitor temporal changes in panicle physiology during the reproductive development. The method is useful for panicle-level measurements under diverse environmental stresses and is sensitive enough to evaluate genotypic variation for panicle physiology and architecture in cereals with compact inflorescences.