Salmonella are among the most important foodborne pathogens and contaminated poultry meat and eggs are the main source of human infection. Infected poultry are a major problem as they are asymptomatic, thus rendering the identification of infected poultry farms difficult. In this context, controlling animal infections is of primary importance. It is known that cell and tissue tropism govern disease in many infectious models, our aim was therefore to identify the infected host-cell types in chicks and the role of the three known bacterial invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutant strains via the intraperitoneal route. Then infected cells in the liver, spleen, gall bladder and aortic vessels were identified using flow-cytometric analyses and invasion confirmed by confocal microscopy. Our results show that all these organs could be foci of infection and that a wide array of phagocytic and non-phagocytic cells is invaded in vivo in each organ. These cells include immune cells and also epithelial and endothelial cells. Moreover, we demonstrated that, despite the invalidation of the three known invasion factors (T3SS-1, Rck and PagN), S. Typhimurium remained able to colonize internal organs and invade non-phagocytic cells in each organ studied. In line with this result, the mutant strains colonized these organs more efficiently than the wild-type strain. S. Typhimurium invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation of chicks and at a level similar to that observed in the spleen. All together, these findings provide new insights into the dynamics of Salmonella spread in vivo in chicks at the organ and cellular levels.Author summaryIn many infectious models, cell and tissue tropism govern disease. Moreover, depending on the entry process, both bacterial behavior and host response are different. It is therefore important to identify the host cells targeted in vivo by a pathogen and the entry routes used by this pathogen to invade the different host cells. This is all the more important with a pathogen that enters cells in several ways like Salmonella, which is responsible for human and animal salmonellosis. As poultry meat and eggs are the main sources of human contamination, controlling animal infections is of primary importance. In this paper, we identified a large array of phagocytic and non-phagocytic cells targeted in vivo using fluorescent Salmonella Typhimurium strains inoculated by the intraperitoneal route. Surprisingly, the same host cells were infected by the wild-type strain or its isogenic mutants deleted of either the T3SS-1 or of the three known invasion factors (T3SS-1, Rck and PagN). These cells were immune cells but also epithelial and endothelial cells. Moreover, we demonstrated for the first time that the gall bladder and the aortic vessels could be foci of infection in chicks in addition to the liver and spleen.