Rising global temperatures have intensified the need to understand molecular mechanisms regulating thermal stress resilience in aquaculture species. This study explores the microRNA (miRNA)-mediated regulatory landscape in Heteropneustes fossilis (Bloch, 1794) exposed to thermal stress, aiming to identify key signatures involved in thermal adaptation. MicroRNAs (miRNAs) are small, non-coding RNAs of approximately 20–24 nucleotides in length that function as critical post-transcriptional regulators of gene expression. and govern adaptation to environmental stressors like temperature fluctuations, hypoxia, salinity, and pollution.Tissue samples were sequenced using the Illumina NovaSeq 6000 platform, yielding high-quality miRNA data with over 180 million total reads. Following rigorous pre-processing using FASTP and Trimmomatic, high-quality reads (15–30 bp) were mapped against the zebrafish (Danio rerio) reference genome and miRBase to identify known and novel miRNAs. De novo prediction via miRDeep2 revealed 313 known and 61 novel miRNAs 61 novel miRNAs with characteristic secondary structures (MFE < –20 kcal/mol; MFEI > 0.8). Differential expression analysis using DESeq2 (p-adj < 0.1; log2FC > |1|) identified significant upregulation and downregulation of key miRNAs in heat-exposed (HE) samples compared to control (HC). Expression data normalization (FPKM) and multivariate analyses including PCA, MA-plots, volcano plots, and heatmaps demonstrated clear clustering and separation of expression profiles, indicating a robust miRNA response to heat stress. Notably, miRNAs targeting stress-related pathways such as PI3K-Akt, MAPK, and HIF-1 signaling were differentially expressed, suggesting their central role in thermal tolerance. These findings provide valuable regulatory insights into the adaptive biology of H. fossilis and offer promising biomarkers for genetic improvement programs. This work contributes to the development of climate-resilient aquaculture strategies by integrating small RNA transcriptomics into functional genomics and breeding frameworks.