Influenza A viruses are characterized by their ability to evade host immunity, even in vaccinated individuals. To determine how prior immunity shapes viral diversity in vivo we studied the intra- and inter-host evolution of equine influenza virus in vaccinated horses. Although the level and structure of genetic diversity was similar to that in na?ve horses, intra-host bottlenecks may be more stringent in vaccinated animals, and mutations shared among horses often fall close to putative antigenic sites.