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. 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) with a four-amino-acid (KRTA) insertion and an I326V mutation (H3 numbering) in the cleavage site of HA to make the motif PEVPKRKRTAR↓GLF. Molecular markers associated with antigenic drift and enhanced pathogenicity in mammals and interspecies transmission were detected in both isolates. Remarkably, both strains gained the F102V and N157D mutations 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, attention should be paid to the importance of continual surveillance of the H7N9 AIVs.