To observe the role of hspA9 of domestic chicken in tolerance of heat stress, hspA9 gene 5 'flanking region of yellow feather dwarf broiler pure line was amplified by PCR and sequenced, and then analyzed by PCR–RFLP. The relationships between the mutation sites detected in hspA9 gene and the five heat resistant traits (triiodothyronine (T3), corticosterone, heterophil/lymphocyte ratio (H/L), CD3+ and CD4+ T cell) were analyzed. The results indicated that three SNP sites (C.–85C>A, C.–485G>A and C.–568G>A) were found. According to the C.–85C>A specific enzyme sites, hspA9 gene is divided into AA, AC and CC genotypes; according to the C.–485G>A specific enzyme sites, this gene is divided into GG, GA and AA genotypes; according to the C.–568G>A specific enzyme sites, this gene is divided into GG, GA and AA genotypes. Under high temperature, C.–485G>A site was highly significantly associated with corticosterone (P＜0.01), corticosterone value in individual of GG genotype was highly significantly higher than that of AA genotype. At room temperature, C.–485G>A site was significantly associated with T3 (P＜0.05), the content of T3 in individual of GA genotype was significantly higher than that of AA genotype; C.–568G>A site was significantly associated with T3 (P＜0.05), the content of T3 in individual of GG genotype was significantly higher than that of AA T3 genotype; C.–568G>A site was significantly associated with CD4+ T cell (P＜0.01), the percentage of CD4+ T cell in individual of GG genotype was significantly higher than that of AA genotype. Both C.+258A>G and C.+276C>G sites play an important role in thermal stress resistance, indicating their potential in marker–assisted selection of chicken.