Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China, and low pathogenic H7N9 viruses have disappeared nationwide. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) based on the sequence of the HA gene. Deduced amino acid sequences of the HA gene revealed that both strains had a four-amino-acid (KRTA) insertion at position 339-342 and an I335V mutation in the cleavage site to make the motif PEVPKRKRTAR↓GLF. Remarkably, both strains gained the F102V and N157D mutations (H3 numbering) in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attentions should be paid to the importance of continual surveillance of the H7N9 AIVs.