In 2004, we isolated triple reassortant H3N2 influenza viruses from turkey breeder hens in Ohio and Illinois. The Illinois flock was vaccinated twice with an inactivated H3N2 vaccine containing a swine origin virus before the outbreak. Additionally, a commercial inactivated vaccine containing an H3N4 virus of duck origin is being used in some turkey breeders. This prompted us to initiate a comparative study on the antigenic and genetic relatedness of various H3 subtype influenza viruses isolated from turkeys, ducks, pigs and humans. The antigenic relatedness between the different viruses was evaluated with the Archetti and Horsfall formula, while nucleotide genetic similarities were calculated using pairwise alignments. Results obtained indicated a high degree of antigenic (>90%) and genetic (>99%) similarities among the turkey-origin H3N2 viruses. However, the turkey viruses were antigenically distantly related to the swine-origin vaccine virus (<30%), although they had approximately 95% genetic similarity in the HA1 gene. Additionally, major genetic and antigenic changes were observed between the turkey viruses and the H3N4 duck vaccine virus as well as the H3N2 human virus. Such genetic and antigenic differences between the turkey-origin viruses and other H3 subtype viruses including vaccine strains could be the reason for the failure in protection in the Illinois turkey breeders vaccinated with swine origin virus. This also emphasizes the importance of using viruses for vaccines that are antigenically similar to the field strains.