In an earlier greenhouse screening, we identified a local indica cultivar HT54 tolerant to high temperature at both seedling and grain-filling stages. In this study, we develop an optimized procedure for fine assessment of this heat tolerance. The results indicated that HT54 seedlings could tolerate high temperature up to 48 °C for 79h. The genetic analysis of F(1) and F(2) offspring derived from the cross between HT54 and HT13, a heat-sensitive breeding line, reveals that the heat tolerance of HT54 was controlled by a dominant major locus, which has been designated as OsHTAS (Oryza sativa heat tolerance at seedling stage). This locus was mapped on rice chromosome 9 within an interval of 420kb between markers of InDel5 and RM7364. The determined candidate ZFP gene has been confirmed to be cosegregated with a single nucleotide polymorphism (SNP) developed PCR-restriction fragment length polymorphism (RFLP) marker RBsp1407 in its promoter region. Another heat tolerance-associated SNP was identified in the first intron of its 5'-untranslated region. The existence of these SNPs thereby indicated that the OsHTAS locus contains at least two alleles. We named the one from HT54 as OsHTAS ( a ) and the one from HT13 as OsHTAS ( b ). Further dynamic expression analysis demonstrated that OsHTAS ( a ) was actively responsive to 45 °C high temperature stress compared with the OsHTAS ( b ) allele.