This study has been aimed to investigate genetic responses for the genes related to heat stress when exposing Holstein calves that were selected based on no significant relationship (inbreeding coefficient <0.01). A total of 10 animals aged from 4 to 6 mo were selected, and heat stress was imposed on animals directly in an environmentally controlled house that was managed for 33°C and 90% of humidity based on THI values. After exposing heat stress for 8 h a day, animals were placed in a normal condition at least 12 h to recover from the heat stress. The blood samples, which were collected at the starting point (09:00 a.m.) and at the end of heat stress (21:00 p.m.), were immediately placed in liquid nitrogen. The RNA was extracted from blood samples between control and treatment, and cDNA library was constructed for each individual. After sequencing analysis that produced a minimum 3 G byte, expression analysis confirmed that 53 genes were differentially expressed according to the severe heat stress. The analysis also verified 4 major pathways (MAPK signaling; T-cell receptor signaling; B-cell receptor signaling, and Chemokine signaling) that were related to heat stress. As expected that heat-shock protein (HSP) is related to thermal stress and response, the analysis verified that HSP70 presented 656.4 fold changes between control and treatments. To verify fold changes, 100 primer pairs from 51 genes with accession numbers were tested using real-time PCR, and the results confirmed 17 genes in statistical significances. The identified genes should be served as reference genes for the selection of superior animals against thermal stress.