Waterfowl play a pivotal role in the evolution of the influenza A virus (IAV), which harbor an extensive repertoire of IAV and act as long-term reservoirs in which low-pathogenic strains can reassort and acquire new genomic segments. In this study, three H1N1 IAVs, named as A/duck/Jiangxi/DP771/2021(H1N1) (DP771), A/duck/DP1433/2021(H1N1) (DP1433), and A/duck/Jiangxi/DP1467/2021(H1N1) (DP1467), were isolated from wild ducks and characterized. Phylogenetic analysis revealed that all these three viruses belonged to Eurasian lineages. In vitro, all these three isolates replicated efficiently in both A549 and MDCK cells, and demonstrated dual receptor binding properties (α2,3- and α2,6-linked sialic acids). Most strikingly, the mouse study showed that two of these three H1N1 viruses, DP1433 and DP1467, replicated efficiently in lungs without pre-adaptation, and DP1433 caused about 20?% bodyweight loss in mice. All these data provide valuable insight into the molecular epidemiology and pathogenicity of duck-original H1N1 viruses circulating in China, and continued surveillance to monitor the diversity of IAVs in ducks is critical to understand the natural history of IAVs and develop efficient strategies against IAVs.