The extreme susceptibility to infection with Salmonella Typhimurium of wild-derived MOLF/Ei mice has been linked to one genomic region on Chromosome 1 (Ity3). A member of the Toll-like receptors family, Tlr5, located on distal Chromosome 1, was previously shown to be a candidate gene for Ity3 based on expression studies and sequencing analysis. The candidacy of Tlr5 as a Salmonella-susceptibility gene was evaluated functionally by comparing Tlr5 C57BL/6J and MOLF/Ei alleles in vitro and in vivo. In vitro studies showed that the MOLF/Ei Tlr5 allele is more transcriptionally active when the gene is removed from its natural genomic environment. This observation was supported by in vivo studies in B6.MOLF-Ity3 congenic mice that showed that mice homozygous for the MOLF/Ei allele at Ity3, including Tlr5, had an increased response to flagellin as measured by IL-6 and CXCL-1 secretion in the serum compared with parental MOLF/Ei mice. Despite the fact that both MOLF/Ei and B6.MOLF-Ity/Ity3 mice are more susceptible to Salmonella Typhimurium infection than B6.MOLF-Ity mice, they exhibit a different phenotype with respect to Tlr5 expression and Tlr5 signaling, supporting the prediction that Tlr5 is not primarily involved in the disease phenotype underlying the Ity3 locus in MOLF/Ei mice.