Salmonella are among the most important foodborne pathogens and contaminated poultry meat and eggs are the main source of human infection. Asymptomatic Salmonella carrier state in poultry renders the identification of infected poultry farms difficult. In this context, controlling animal infections is of primary importance. In many infectious models, cell and tissue tropism govern disease. 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. Our aim was thus to identify the host-cell types infected in chicks and to determine the role in this process of the three Salmonella invasion factors known today: the type III secretion system T3SS-1 and the two outer membrane proteins Rck and PagN. After intracelomic inoculation of chicks, the fluorescent wildtype S. Typhimurium 14028 strain and its isogenic mutant deleted of the three know invasion factors were detected using flow-cytometry. Cell invasion was confirmed by confocal microscopy. Our results show that phagocytic and non-phagocytic cells, including immune cells, epithelial and endothelial cells, are invaded by both the wild-type and the mutant strain in vivo in the gall bladder, liver, spleen and vessels. In line with these results, mutants defective for the T3SS-1 or the three known invasion factors, better colonized chicks than their wild-type parent strain. All together, these findings provide new insights into the dynamics of Salmonella spread in vivo in chicks at the organ and cellular levels.