With the increasing global warming, high temperature seriously threatens rice production and food security. Identifying heat-tolerant genes and cultivating new heat-resistant varieties are crucial approaches to deal with the harm of high temperature. A set of RIL population, derived from the cross between the heat-sensitive variety Junambyeo(JNB) and the strong heat-tolerant variety Ganzaoxian58(GZX58), identified the heat tolerance related traits and QTL at heading stage under extreme natural high-temperature conditions based on high-density genetic map.Under high-temperature stress, seed setting rate was significantly reduced, which is significantly correlated with the control seed setting rate and heat resistance coefficient. The RIL population''s seed-setting rates under high temperature showed a continuous and approximately normal distribution, exhibiting highly significant correlations with control seed-setting rates and heat tolerance coefficients.Six QTL were detected for spikelet number, filled grain number and seed setting rate under control environment, five heat tolerance QTL were detected under high temperature stress, including qHTSF4, qHTSFII4, qHTC2, qHTC4 and qHTC6, among these QTLs qHTSFI4, qHTSFII4 and qHTC4 were located in the same chromosome region, forming a major QTL cluster qHTH4. Utilizing high-density genetic map, extreme lines genotype and bioinformatics, it was confirmed that qHTC2, qHTH4 and qHTC6 function in regulating heat tolerance and had additive effects, in here, the RIL lines that aggregated more than two major QTL had significantly enhanced heat resistance, and then 14 directly related gene were screened in the key QTL mapping target regions, which laid the foundation for the genetic improvement of heat tolerance and molecular mechanism analysis of key genes in rice.