In vivo bioluminescence imaging facilitates the non-invasive visualization of biological processes in living animals. This system has been used to track virus infections mostly in mice and ferrets; however, until now this approach has not been applied to pathogens in avian species. To visualize the infection of an important avian pathogen, we generated Marek's disease virus (MDV) recombinants expressing firefly luciferase during lytic replication. Upon characterization of the recombinant viruses in vitro, chickens were infected and the infection visualized in live animals over the course of 14 days. The luminescence signal was consistent with the known spatiotemporal kinetics of infection and the life cycle of MDV, and correlated well with the viral load measured by qPCR. Intriguingly, this in vivo bioimaging approach revealed two novel sites of MDV replication, the beak and the skin of the feet covered in scales. Feet skin infection was confirmed using a complementary fluorescence bioimaging approach with MDV recombinants expressing mRFP or GFP. Infection was detected in the intermediate epidermal layers of the feet skin that was also shown to produce infectious virus, regardless of the animals' age at and the route of infection. Taken together, this study highlights the value of in vivo whole body bioimaging in avian species by identifying previously overlooked sites of replication and shedding of MDV in the chicken host. Author summary: In vivo bioluminescence imaging is a powerful tool to track virus infection in the whole body of living animals. This system has been successfully used in mice, ferrets, rats and even fishes, but until now never in birds. In this study, we performed the first in vivo imaging assessing the spread of an important avian pathogen, the highly oncogenic Marek's disease virus (MDV). Using a recombinant virus expressing firefly luciferase, we visualized the course of MDV infection in chicks for 14 days. The bioluminescent signal was consistent with the known kinetics and sites of dissemination of MDV, notably in feathers. With this new approach, we also discovered two novels sites of early infection and replication that may contribute to persistent virus shedding. Both novel sites represent hard skin appendages like the feathers: the beak and the skin of the feet that are covered in scales. These results were confirmed with two recombinant viruses expressing fluorescent proteins. Fifty-five years after the discovery of MDV and thanks to in vivo imaging, we provide new insights in MDV life cycle in vivo, highlighting the importance of bioluminescence imaging of the entire body in living animals. [ABSTRACT FROM AUTHOR]