<p>Environmental heat stress is a major concern in livestock production especially in water buffalo, which are more sensitive to heat stress. Buffalo bulls experiencing heat stress exhibit reduced feed intake and semen production, triggering stress responses at multiple levels. This comprehensive study delves into the understanding of the complex nature of heat stress responses in Murrah bulls through gene expression analysis. Murrah bulls were categorized into a seasonally non-affected (<i>n</i> = 6) and seasonally affected (<i>n</i> = 6) groups, on the basis of semen quality in different seasons, classified as hot summer, comfort and winter. The study has revealed that heat stress having an impact on physiological variables including scrotal temperature and respiration rate as well as serum antioxidants levels. Additionally, the expression of genes in response to heat stress and oxidative stress was analyzed in peripheral blood mononuclear cell of the categorized bulls. The study also includes expression analysis of targeted semen quality genes in spermatozoa of the target bulls. The seasonal fluctuations have been noticed to have an influence on the physiological, biochemical, and molecular levels among the bulls with varying semen quality under heat stress. Significant differences were observed in the scrotal infrared thermography and expression of heat shock proteins as well as leptin gene in the both groups, specifically during the summer season. The outcome of this study contributes to the holistic understanding of heat stress resilience in Murrah buffalo bulls and provides foundation for future strategies to improve their well-being and better reproductive efficiency under heat stress conditions.</p> Graphical Abstract <p></p>