Rice (Oryza sativa L.) is sensitive extremely to heat stress at the flowering stage. Screening for rice genetic resources with heat tolerance at the flowering stage and identifying the quantitative trait locus (QTL) become important on the improvement of heat tolerance. In this study, 205 rice lines were evaluated for heat tolerance in the field. The rice panicles which flowered under high temperature were tagged for quantifying the spikelet fertility (SF) which was subjected for the genome wide association study(GWAS). The broad variation on the spikelet fertility was observed in heat tolerance among rice accessions, which ranged from 19.0% to 86.6%, with an average of 64.0% and a median of 65.9%. Rice lines such as “06-32”, “Jiandaoqi” and “Louzaoxian No.5” were tolerant against heat stress and they could be used as candidate donor lines in future rice breeding. A total of 18 QTL with 130 SNPs were detected to be significantly associated with heat tolerance. Six QTL were co-localized with the loci which were previously identified. A novel QTL, qHT4-6, with the lowest P-value was selected for the allelic variation analysis. The heat tolerance of rice lines with G-allele of the lead SNP were significantly stronger than those of A-allele lines. As a result, six genes within the interval of qHT4-6 were proposed to be candidate responding to the heat stress.