In this Research Communication we describe the effect of temperature and humidity index (THI) on various physiological traits, the plasma heat shock protein 70 (HSP70), heat shock protein 90 (HSP90) and cortisol levels and other blood parameters in crossbred buffalo (Nili-Ravi × Murrah) and Mediterranean buffalo to compare their tolerance to heat stress. As expected, crossbred buffalo had a significantly higher rectal temperature (RT), body surface temperature (BT), respiratory rate (RR), HSP70 and HSP90 levels in summer compared to spring and winter. RT and BT were also significantly higher in spring compared to winter. A significant correlation existed between THI and RT (r = 0·81) and RR (r = 0·84). Importantly, in summer the crossbred buffalo had a significantly lower RT, BT and RR and higher HSP70, HSP90 and cortisol levels than the Mediterranean buffalo. In conclusion, higher THI was associated with significant increase in RT, RR, BT, HSP70, HSP90 and cortisol levels, and the crossbred buffalo were more heat tolerant than Mediterranean buffalo.