| Summary: | Influenza A virus (IAV) is widespread in wild bird reservoirs. Sixteen hemagglutinin subtypes are associated with wild waterfowl hosts; some subtypes are isolated infrequently, one of which is H12 IAV. In this study, we detected three H12 IAVs from <i>Anas</i><i>crecca</i> and <i>Anas formosa</i> in Poyang Lake, China, in 2018, one of which was isolated. Phylogenetic analysis revealed that the genome sequences of the three H12 viruses belonged to the Eurasian lineage, except for PA genes and one NP gene, which belonged to the North American lineage. The growth kinetics showed that the H12 isolate grew better in A549 than MDCK cells. Moreover, although the H12 isolate cannot efficiently replicate in BALB/c mice, it can bind to both <i>α</i>-2,6 sialic acid (SA) and <i>α</i>-2,SA-linked receptors. In addition, we examined the phylodynamics of H12 viruses by Bayesian phylogeographic analysis. The results show that two major transmission routes of H12 IAVs were from Asia to Oceania and from Europe to South America, and <i>Anas</i> and <i>Arenaria</i> genera were the major hosts of the viral transmission. Our findings help us better understand the evolution of H12 IAV and highlight the need for the continued surveillance of IAVs circulating in wild birds.
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