This study was conducted on 30 freshly parturated multiparous crossbred dairy cows possessing three levels of Holstein Frisian genetic makeup (62.5, 75.0, and 87.5%). Data on temperature humidity index (THI) were classified into comfortable (≤ 71), mild stress (72–79), moderate stress (80–89), and stressful (≥90) zone. Results showed that serum cortisol concentration increased significantly (P < 0.05) in cows during stressful condition irrespective of genetic makeup compared to the other zones. Daily milk yield (DMY) was significantly (P < 0.05) lower in cows during stressful condition. Triglyceride was significantly higher in cows with genetic makeup 87.5% compared to the others, while total serum protein was significantly (P < 0.05) higher in cows during both moderate and stressful conditions. The mean concentration of cortisol and protein increased linearly from comfort to the stressful condition, while mean serum triglyceride, glucose, progesterone (P4), and luteinizing hormone (LH) decreased by moving from comfort to stressful conditions. Results also indicated that higher cortisol level in higher grade crossbred cows was adversely associated with LH concentration and milk yield under thermal stress conditions. Greater triglyceride in high-grade crossbred (87.5%) cows indicates higher fat mobilization reflecting a negative energy balance. We concluded that heat stress increased blood cortisol and protein, and reduced milk yield in dairy cows irresptive of the genetic makeup. In addition, there was no significant difference in blood metabolites and daily milk yield in the different levels of genetic makeup cows.