introduction: Heat stress poses a significant challenge to the dairy sector, leading to substantial losses due to its detrimental effects on both milk production and reproductive performance. In response to high temperatures, cows have developed various protective mechanisms to mitigate the adverse consequences of heat stress. Some cows maintain their productive and reproductive capabilities during hot summer conditions and are considered thermotolerant, making them potential candidates for selective breeding programs aimed at enhancing heat resilience. Methods: In the present study, we conducted a comprehensive analysis by collecting samples from both thermotolerant and thermosensitive cows, employing a whole-genome sequencing approach to elucidate the unique genetic variants that distinguish the two groups. After identifying group-specific variants, we focused on variants within miRNA genes using miRNASNP v4. Results: We successfully identified 15 variants in thermotolerant cows, including 1 novel variant, across 15 distinct miRNAs. Five of these variants were located in mature miRNAs, potentially disrupting their structure and functionality. Subsequent analyses (Gene Ontology and KEGG) indicated that several of these variants could also influence miRNA-mRNA interactions, thereby impacting various molecular pathways, including metabolic pathways, the MAPK signaling pathway, the Ras signaling pathway, and mitophagy. Conclusions: This research provides valuable insights into the underlying mechanisms associated with thermotolerance in dairy cattle. Understanding the role of these miRNA variants may lead to innovative approaches in livestock management, ultimately contributing to improved animal welfare and productivity under changing climatic conditions.