Toll-like receptor 5 (TLR5) is activated by bacterial flagellins and plays a crucial role in the first-line defence against pathogenic bacteria and in immune homeostasis, and is highly conserved in vertebrate species. However, little comparative information is available on TLR5 functionality. In this study, we compared TLR5 activation using full-length and chimeric TLR5 of various vertebrate species (human, chicken, mouse, pig, cattle). Chimeric TLR5 receptors, consisting of human transmembrane and intracellular domains, linked to extracellular domains of animal origin, were generated and expressed. The comparison of chimeric TLR5s and their full-length counterparts revealed significant functional disparities. While porcine and chicken full-length TLR5s showed a strongly reduced functionality in human cells, all chimeric receptors were functional when challenged with TLR5 ligand Salmonella FliC. Using chimeric receptors as a tool allowed for the identification of ectodomain-dependent activation potential and partially host species-specific differences in response to various enteric bacterial strains and their purified flagellins. We conclude that both the extra- and intracellular determinants of TLR5 receptors are crucial for compatibility with the species expression background and hence for proper receptor functionality. TLR5 receptors with a common intracellular domain provide a useful system to investigate bacteria- and host-specific differences in receptor activation.