Salmonella enterica serovar Enteritidis is the predominant serovar associated with salmonellosis worldwide, which is in part due to its ability to contaminate the internal contents of the hen's egg. It has been shown that S. enterica serovar Enteritidis has an unusual tropism for the avian reproductive tract and an ability to persist in the oviduct and ovary. Factors allowing S. enterica serovar Enteritidis strains to contaminate eggs could be a specific interaction with the oviduct tissue, leading to persisting oviduct colonization. In vivo expression technology, a promoter-trap strategy, was used to identify genes expressed during oviduct colonization and egg contamination with S. enterica serovar Enteritidis. A total of 25 clones with in vivo-induced promoters were isolated from the oviduct tissue and from laid eggs. Among the 25 clones, 7 were isolated from both the oviducts and the eggs. DNA sequencing of the cloned promoters revealed that genes involved in amino acid and nucleic acid metabolism, motility, cell wall integrity, and stress responses were highly expressed in the reproductive tract tissues of laying hens.