Heat stress is one of the most critical environmental factors impacting rice cultivation, driven by the rising global temperatures. Therefore, understanding the differences in molecular mechanisms of heat stress tolerance between rice cultivars, particularly indica and japonica, is crucial for developing heat-tolerant varieties. In this study, high throughput RNA-sequencing technology was utilized to explore the transcriptomic changes in the root tissues of two rice varieties, 93-11 (indica) and ZH11 (japonica) under heat stress and during recovery. Differentially Expressed Genes (DEGs) analysis revealed that ZH11 had 14,719 DEGs after the two-day heat treatment, and 10,178 DEGs during the recovery process. In contrast, 93-11 had a lower number of DEGs than ZH11 in both the heat treatment and recovery phases, with 12,433 DEGs and 5986 DEGs, respectively. The GO and KEGG analyses showed that the two rice varieties shared several enriched pathways in response to heat stress. However, each cultivar also had its own uniquely enriched pathways, reflecting distinct responses to heat stress in ZH11 and 93-11. In addition, WGCNA analysis highlighted that the OsMAPK3 is novel hub gene in response to heat stress in rice. Knockout of OsMAPK3 compromises rice heat stress tolerance. These results provide new insights into the differences in molecular mechanisms of heat stress response in roots between indica and japonica rice cultivars, offering valuable targets for genetic improvement and breeding programs aimed at developing heat-tolerant rice varieties.