At present, avian-origin H3N2 subtype canine influenza virus (H3N2 CIV) is prevalent in East Asian and North American countries. The host tropism of H3N2 CIV to mammals, including mice, guinea pigs, ferrets, and pigs, has been evaluated. However, it has not previously been determined whether avian-origin H3N2 CIV can be transmitted back to birds. In China, ducks, chickens, geese, pigeons, and quails are economically important domestic poultry that are susceptible to numerous subtypes of influenza A virus. These poultry occasionally or frequently come into contact with dogs. In this study, the infectivity of the first- and last-isolated Chinese H3N2 CIV strains in these poultry species was evaluated, and oropharyngeal and cloacal swabs of these animals were negative for virus, as determined by specific pathogen-free (SPF) chicken egg inoculation and real-time RT-qPCR assays. Clinical signs and gross lesions were not observed in any of these species, and seroconversion also did not occur. The results showed that all these avian species were unsusceptible to the first- and last-isolated H3N2 CIVs, indicating unidirectional evolution of the mammalian host tropism of H3N2 CIV.