Molecular chaperones have been understood to be preferentially transcribed to prevent perturbations in response to various stresses. In this study, three single nucleotide polymorphisms (SNPs), g.324G>C, g.589C>T and g.651C>G in Heat shock factor binding protein 1 (HSBP1) gene were found and genotyped in 930 Chinese Holstein cattle. The results indicated that only g.589C>T polymorphism locus met Hardy-Weinberg equilibrium (P > 0.05). Pair linkage disequilibrium analysis and haplotype construction of HSBP1 gene were performed using SHEsis software. Seven haplotypes were constructed and fourteen haplotype combinations were found. Association analysis showed that H2H2 haplotype combination was advantageous for thermo tolerance breeding in Chinese Holstein. The cows with H2H2 haplotype combination have lower decrease rate of milk yield than those with H2H3 haplotype combination (P < 0.05) and lower potassium content in erythrocytes (PCE) than those with H2H5 (P < 0.05), H4H4 (P < 0.05) and H4H5 (P < 0.01) haplotype combination. The association between SNP and thermo tolerance traits showed that PCE of cows with GG genotype was lower than those with CG genotype at g.651C>G locus (P < 0.01). Pair linkage disequilibrium analysis revealed that the three loci were at a strong disequilibrium state. So we presumed that the effect of H2H2 haplotype combination on thermo tolerance traits major due to the SNP of g.651C>G.