Entamoeba histolytica (E. histolytica) exhibits a remarkable capacity to respond to thermal shock stress through a sophisticated genetic regulation mechanism. This process is carried out via Heat Shock Response Elements (HSEs), which are recognized by Heat Shock Transcription Factors (EhHSTFs), enabling fine and precise control of gene expression. Our study focused on screening for HSEs in the promoters of the E. histolytica genome, specifically analyzing six HSEs, including Ehpgp5, EhrabB1, EhrabB4, EhrabB5, Ehmlbp, and Ehhsp100. We discovered 2578 HSEs, with 1412 in promoters of hypothetical genes and 1166 in coding genes. We observed that a single promoter could contain anywhere from one to five HSEs. Gene ontology analysis revealed the presence of HSEs in essential genes for the amoeba, including cysteine proteinases, ribosomal genes, Myb family DNA-binding proteins, and Rab GTPases, among others. Complementarily, our molecular docking analyses indicate that these HSEs are potentially recognized by EhHSTF5, EhHSTF6, and EhHSTF7 factors in their trimeric conformation. These findings suggest that E. histolytica has the capability to regulate a wide range of critical genes via HSE-EhHSTFs, not only for thermal stress response but also for vital functions of the parasite. This is the first comprehensive study of HSEs in the genome of E. histolytica, significantly contributing to the understanding of its genetic regulation and highlighting the complexity and precision of this mechanism in the parasite’s survival.