Control of coccidiosis in broiler chickens continues to pose challenges to commercial poultry producers, especially in an era of increased consumer demand for antibiotic-free broiler production.As a result, coccidiosis vaccines are now commonlyused in rotation programs to achieve effective coccidiosis control. Inovocox EM1 vaccine (EM1) is a coccidiosis vaccine that allows for earlier immune acquisition through oocyst cycling, which reduces theeffects of wild-type coccidia. The EM1 vaccine is administered to embryonated broiler hatching eggs between18 and 19 D of incubation (doi). In the U.S., commercial broiler hatcheries vaccinate embryonated eggs ateither 18.5 or 19 doi. However, it is unclear whether adifference in embryo age at the time of in ovo injectioncan impact the actual site of vaccine delivery. In addition, it is unclear where oocysts eventually become localized within the embryo following the in ovo injectionof EM1. Therefore, the objective of this study was todetermine the effects of stage of embryonic developmenton the actual deposition site of the EM1 vaccine oocystswhen they are in ovo injected and to subsequently investigate the movement and eventual location of EM1oocysts after in ovo injection. Because all eggs were injected at the same time, a 12-h difference in set time wasa means to derive 18.5 and 19.0 incubation age of injection (IAN) treatments. The experimental design wasa 3 injection treatment (noninjected, diluent-injected,and vaccine-injected) × 2 IAN factorial. There was asignificant main effect of IAN on site of vaccine oocystsdelivery, and subsequent hatching chick quality. Qualitative histological evaluation revealed the oral uptakeof vaccine oocysts through the amnion, with their subsequent presence in the gizzard and intestinal lumenby 24 to 36 h postinjection. In conclusion, physiological development influenced the site of injection, andoocysts imbibed along with the amniotic fluid in latestage broiler embryos are subsequently transported tothe gastrointestinal tract