Toll-like receptors (TLR) recognize evolutionarily conserved molecular motifs (pathogen-associated molecular patterns) of infectious microbes and initiate innate immune response upon activation with relevant pathogens. This study investigated the acute effect of Salmonella Enteritidis challenge on TLR mRNA expression in cecum and spleen of birds from 3 distinct genetic lines. Chicks from broiler, Leghorn, and Fayoumi lines were inoculated or mock-inoculated with Salmonella Enteritidis. The mRNA expression levels of TLR2, TLR4, and TLR5 genes were assessed by quantitative reverse transcription-PCR of cecum and spleen tissue harvested at 2 or 18 h postinoculation (PI). There were no significant genetic line effects on TLR mRNA expression in spleen or cecum of mock-infected birds, or in the cecum of infected birds. Genetic line effect was significant (P < 0.05) on TLR mRNA expression in the spleen of Salmonella Enteritidis- infected birds. The Fayoumi line had higher TLR2 and TLR4 expression than Leghorn, higher TLR2 mRNA expression than broiler, and the broiler line had higher TLR5 expression than Leghorn and Fayoumi. In Salmonella Enteritidis-infected birds, the TLR2 expression in both cecum and spleen and TLR4 expression in spleen were significantly higher at 18 h PI than 2 h PI. The results demonstrate a significant genetic line effect on TLR expression in the spleen of Salmonella Enteritidis-infected birds, which may partly explain genetic variability in immune response to Salmonella Enteritidis.