From 2013 to 2015, 32 H1-subtype avian influenza viruses (AIVs), H1N2 (n = 12), H1N3 (n = 14), H1N4 (n = 4) and H1N9 (n = 2), were isolated from poultry in Zhejiang Province in eastern China. These strains were characterized by whole-genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that these strains clustered in the AIV Eurasian lineage. These strains were found to be minimally pathogenic in mice and were able to replicate in mice without prior adaptation. Continued surveillance is needed, considering the important role of poultry in AIV reassortment.