BackgroundHeat stress threatens rice yield and quality at flowering stage. In this study, average relative seed setting rate under heat stress (RHSR) and genotypes of 284 varieties were used for a genome-wide association study.ResultsWe identified eight and six QTLs distributed on chromosomes 1, 3, 4, 5, 7 and 12 in the full population andindica, respectively.qHTT4.2was detected in both the full population andindicaas an overlapping QTL. RHSR was positively correlated with the accumulation of heat-tolerant superior alleles (SA), andindicaaccession contained at least two heat-tolerant SA with average RHSR greater than 43%, meeting the needs of stable production and heat-tolerant QTLs were offer yield basic for chalkiness degree, amylose content, gel consistency and gelatinization temperature. Chalkiness degree, amylose content, and gelatinization temperature under heat stress increased with accumulation of heat-tolerant SA. Gel consistency under heat stress decreased with polymerization of heat-tolerant SA. The study revealedqHTT4.2as a stable heat-tolerant QTL that can be used for breeding that was detected in the full population andindica. And the grain quality ofqHTT4.2-haplotype1 (Hap1) withchalk5,wx, andalkwas better than that ofqHTT4.2-Hap1 withCHALK5,WX, andALK.Twelve putative candidate genes were identified forqHTT4.2that enhance RHSR based on gene expression data and these genes were validated in two groups. Candidate genesLOC_Os04g52830andLOC_Os04g52870were induced by high temperature.ConclusionsOur findings identify strong heat-tolerant cultivars and heat-tolerant QTLs with great potential value to improve rice tolerance to heat stress, and suggest a strategy for the breeding of yield-balance-quality heat-tolerant crop varieties.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04260-5.