Heat shock proteins (HSPs) play a crucial role in cellular protection during environmental stress, particularly in aquatic organisms sensitive to temperature fluctuations and pollution. In this study, we conducted an in silico analysis of gene expression profiles of key HSP genes (HSP70, HSP90, and HSP27) in selected Indian freshwater fish species. Public gene expression data were retrieved from the NCBI Gene Expression Omnibus (GEO) and analyzed using GEO2R and other bioinformatics tools. We observed significant upregulation of HSP70 and HSP90 under thermal stress and pollutant exposure, with HSP27 showing variable expression. Phylogenetic analysis of HSP70 sequences across species was performed using MEGA11, revealing evolutionary relationships and conserved motifs. These findings underline the adaptive molecular response of freshwater fishes to environmental stress, highlighting the utility of HSP genes as potential biomarkers for aquatic health monitoring and conservation biology.