This study examined the potential for cross-species transmission of influenza viruses by comparing the genetic and pathogenic characteristics of H1 avian influenza viruses (AIVs) with different host origins in Korea. Antigenic and phylogenetic analyses of H1 AIVs circulating in Korea provided evidence of genetic similarity between viruses that infect domestic ducks and those that infect wild birds, although there was no relationship between avian and swine viruses. However, there were some relationships between swine and human viral genes. The replication and pathogenicity of the H1 viruses was assessed in chickens, domestic ducks, and mice. Viral shedding in chickens was relatively high. Virus was recovered from both oropharyngeal and cloacal swabs up to 5-10 days post-inoculation (p.i.). The titers of domestic duck viruses in chickens were much higher than those of wild bird viruses. Both domestic duck and wild bird viruses replicated poorly in domestic ducks. None of the swine viruses replicated in chickens or domestic ducks; however, six viruses showed relatively high titers in mice, regardless of host origin, and induced clinical signs such as ruffled fur, squatting, and weight loss. Thus, although the phylogenetic and antigenic analyses showed no evidence of interspecies transmission between birds and swine, the results suggest that Korean H1 viruses have the potential to cause disease in mammals. Therefore, we should intensify continuous monitoring of avian H1 viruses in mammals and seek to prevent interspecies transmission.