Toll-like receptors (TLR) recognize pathogenassociated molecular patterns (PAMP) of infectious microbes. Activation of TLR with PAMP can result in immune response by modulation of innate and adoptive immune system. This study aimed to investigate the acute effect of Salmonella challenge on TLR RNA expression in cecum and spleen of birds from different genetic lines. Chicks from broiler, Leghorn, and Fayoumi lines were challenged or mock challenged with Salmonella. The RNA expression levels of TLR2, TLR4 and TLR5 genes were assessed by quantitative RT-PCR in cecum and spleen tissue harvested at 2 or 18 h post-challenge. The results demonstrate a significant genetic line effect on TLR expression in the spleen of Salmonella infected birds, which may partly explain the genetic variability in immune response to Salmonella enterica serovar Enteritidis. The higher level of TLR2 and TLR4 RNA expression observed in the spleen of Fayoumi line compare to Leghorn and broiler lines in Salmonella enterica serovar Enteritidis challenged birds may be associated with the stronger immune response to the infection and might be useful characteristics to be considered in breeding immunocompetent chickens.