<p>Temperature is a key abiotic stressor that influences the growth and survival of aquatic organisms. Hilsa Shad is an anadromous fish of significant socio-economic importance in South Asia, including Bangladesh. Due to the migratory nature, it faces continuous temperature fluctuations across different habitats. The expression of Heat shock protein 60 (Hsp60) is closely correlated with responses to environmental challenges, particularly temperature-induced stress. Up to date, no studies have investigated the response of Hilsa Shad to heat stress during its migration in a specific season or time frame. Therefore, in this study, a genome-wide search of the Hilsa Shad was operated by using the NCBI-BLAST program and identified three isoforms of the Hsp60 gene. The fish samples were collected from freshwater, brackish, and marine habitats in mid-February, which is the period of their downstream migration. Specific RT-qPCR primers were designed to investigate the relative expression of Hsp60 isoforms in gill, kidney, liver, and muscle tissue aiming to reveal their thermal responses across habitats. The expression results showed that all three isoforms exhibited varying levels of expression among these habitats for all of the investigated tissues. For the first time since the whole genome sequencing of Hilsa Shad, this study explored the genomic basis of how Hilsa adapts to temperature stress across variable habitats during migration. These findings contributed to a better understanding of stress-responsive gene regulation in this commercially important species. This work holds potential implications for climate-resilient fisheries and sustainable Hilsa aquaculture amid changing environmental conditions.</p>