Heat stress inhibits rice panicle development and reduces the spikelet number per panicle. This study investigated the mechanism involved in heat-induced damage to panicle development and spikelet formation in rice cultivars that differ in heat tolerance. Transcriptome data from developing panicles grown at 40°C or 32°C were compared for two rice cultivars: heat-tolerant Huanghuazhan and heat-susceptible IR36. Of the differentially expressed genes (DEGs), 4,070 heat stress-responsive genes were identified, including 1,688 heat-resistant-cultivar-related genes (RHR), 707 heat-susceptible-cultivarrelated genes (SHR), and 1,675 common heat stress-responsive genes. A Gene ontology (GO) analysis showed that the DEGs in the RHR category were significantly enriched in 54 gene ontology terms, some of which improved heat tolerance, including those in the WRKY, HD-ZIP, ERF, and MADS transcription factor families. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs in the RHR and SHR categories were enriched in 15 and 11 significant metabolic pathways, respectively. Improved signal transduction capabilities of endogenous hormones under high temperature seemed to promote heat tolerance, while impaired starch and sucrose metabolism under high temperature might have inhibited young panicle development. Our transcriptome analysis provides insights into the different molecular mechanisms of heat stress tolerance in developing rice.