Heat shock transcription factor (Hsf) plays a pivotal role in regulating plant growth, development, and stress responses. Hsf activates or represses target gene transcription by binding to the heat shock element (HSE) of downstream genes. However, the specific interaction sites between Hsf and the HSE in the promoter remain unclear. In this study, the critical amino acid residues of ZmHsf17 and the paralogous ZmHsf05 involved in DNA binding were identified using molecular docking models, site-directed mutagenesis, and the electrophoretic mobility shift assay (EMSA). The results reveal that both ZmHsf17 and ZmHsf05 bind to the HSE of the promoter via a conserved arginine residue located in the α3 helix of their DNA-binding domains. Sequence substitution experiments among distinct HSEs demonstrated that flanking sequences upstream and downstream of the HSE core synergistically contribute to the specificity of DNA-binding domain recognition. Comparative evolutionary analysis of DNA-binding domain sequences from 25 phylogenetically diverse species reveals that the α3 helix constitutes the most conserved structural element. This study elucidates the key interaction sites between maize HsfA2 and its target genes, providing theoretical insights into the binding specificity to the HSEs of the plant's Hsf family and the functional divergence. Additionally, these findings offer new targets for the precise engineering of Hsf proteins and synthetic HSEs.