The Arkansas-type (Ark) IBV has continued to be the most commonly isolated serotype in commercial poultry for the past two decades in the United States. Experimental and field trials that used the live-attenuated Ark vaccine, Ark Delmarva Poultry Industry (ArkDPI), have shown that it exhibits poor replication in chickens and confers poor protection against challenge. Re-isolation of the ArkDPI virus from vaccinated birds has shown that certain polymorphisms arise and are selected for upon infection and replication in birds. Most of the polymorphisms observed are located in the S1 spike protein gene, suggesting that S1 spike is an important factor that determines virus infectivity and replication. Previous studies have shown that ArkDPI administered by the eye drop route achieves an adequate level of infection and replication in birds. When ArkDPI is delivered by spray however, infection and replication in birds is poor. We increased the doses of ArkDPI delivered by spray and observed that a 100x dose achieved the same level of infection and replication as ArkDPI administered by eye drop. Sequencing of the S1 spike gene from re-isolated from vaccinated birds showed the certain polymorphisms emerge as expected. Two polymorphisms most commonly observed in re-isolated ArkDPI vaccine viruses are a tyrosine to histidine change in position 43 (Y43H) and a deletion of an arginine residue at position 344 (344). The S1 spike protein is responsible for attachment to host cells and is a primary determinant of tropism and thus, changes in the S1 spike protein sequence could affect the virus ability to bind to host tissues. To examinethis, we cloned, expressed, and purified ArkDPI S1 spike proteins carrying Y43H and/or 344 changes and used recombinant S1 spike proteins in a protein histochemistry assay on trachea from mature birds and chorioallantoic membrane (CAM) tissues from embryonated eggs. We observed that the Y43H change enhanced binding to trachea, whereas the original ArkDPI vaccine spike (H43) had the highest binding to CAM. Western blot of recombinant S1 spike proteins showed that ArkDPI serotype-specific antisera was unable to recognize linear epitopes on S1 spike proteins with 344, suggesting that 344 change alters the antigenicity of the ArkDPI S1 spike. Taken together, these results demonstrate the emergence of polymorphisms in re-isolated ArkDPI vaccine viruses from chickens can be explained by the selective advantage offered by enhanced binding to host tissues, as in the case for Y43H; and by alterations in antigenicity, as in the case of 344. Furthermore, the viruses with such polymorphisms appear to be in low numbers in the ArkDPI vaccine preparation and for this reason, rates of infection and replication are low upon spray vaccination with ArkDPI.