Toll-like receptor 7 (TLR7) is critical for the triggering of innate immune response by recognizing the conserved molecular patterns of single-stranded RNA (ssRNA) viruses and mediated antigenic adaptive immunity. To understand how TLR7 distinguish pathogen-derived molecular patterns from the host self, it is essential to be able to identify TLR7 receptor interaction interfaces, such as active sites or R848-agonist binding sites. The functional interfaces of TLR7 can serve as targets for structure-based drug design in studying the TLR7 receptor’s structure-function relationship. In contrast to mammalian TLR7, chicken TLR7 (chTLR7) is unknown for its important biological function. Therefore, it has been targeted to mediate contrasting evolutionary patterns of positive selection into non-synonymous SNPs across eleven species using TLR7 conservation patterns (evolutionary conserved and class-specific trace residues), where protein sequence differences to the TLR7 receptors of interest record mutation that have passed positive section across the species. In this study, we characterized the Lys609 residue on chTLR7-ECD homodimer interfaces to reflect the current tendency of evolving positive selection to be transfer into a stabilization direction of the R848-agonist/ chTLR7-ECDs complex under the phylogenetically variable position across species and we suggest a potential indicator for contrasting evolutionary patterns of both the species TLR-ECDs.