Poultry products are the major source of food-borne Salmonella infection in humans. Broiler lines selected to be more resistant to Salmonella could reduce the transfer of Salmonella to humans. To investigate differences in the susceptibility of newly hatched chicks to oral infection with Salmonella enterica serovar Enteritidis, 3 commercial broiler lines (A, B, and C) were infected immediately after hatch and compared to healthy controls at 0.33, 1, and 2 d postinfection. Weight, bacteriological examination, and the jejunal influx of CD4, CD8, TCRαβ, TCRγδ, and KUL01 (macrophages and dendritic cells) cells that are positive was investigated. In addition, the jejunal transcriptional response was analyzed using whole-genome chicken cDNA arrays. Salmonella colony-forming unit counts from cecal content and liver revealed that Salmonella enterica entered the body at 0.33 d postinfection. Broiler line A appeared most susceptible to intestinal colonization and the systemic spread of Salmonella. In addition, the Salmonella-induced jejunal influx of macrophages in this line showed a clear increase in time, which is in contrast to lines B and C. On the other hand, all lines showed a peak of CD4+ cells at 1 d postinfection when infected chicks were compared to control chicks. The transcriptional response of line A clearly differed from the responses in lines B and C. Functional analysis indicated that the majority of the differentially expressed genes at 0.33 d postinfection in line A were involved in cell-cycle functions, whereas at 2 d postinfection the majority of the differentially expressed genes could be assigned to inflammatory disorder, differentiation and proliferation of (T) lymphocytes. These data indicate that hatchlings of different broiler lines differ in their systemic spread of Salmonella and suggest that intestinal barrier functions, as well as immunological responses, may be the underlying factors. We hypothesize that the differences between genetic chicken lines divergent in their response to Salmonella infection at a young age include developmental differences of the gut.