Abstract:[Objective] To analysis the molecular genetic characteristics of influenza A virus H3N2 and N7N9 viruses in one case of mixed infection. [Methods] Using a set of primers and probes for influenza virus genotyping, specimens were detected by real-time quantitative PCR method. Whole-genome sequences of virus isolates were obtained by second-generation sequencing technology. [Results] In April 2013, one mixed infection with seasonal H3N2 influenza virus and avian influenza H7N9 virus were confirmed in Nanjing, and two mixed viruses were isolated, which named A/Nanjing/M1/2013 (H3N2) (M1-H3N2) and A/Nanjing/M2/2013 (H7N9) (M2-H7N9), respectively. Some important molecular markers associated with host adaptation and virulence were identified in M2-H7N9 virus. M2-H7N9 virus had substitution Q226L (H3 numbering) in the receptor-binding site of hemagglutinin, which may facilitate the viruses to bind to sialic acid (SA)-2,6-Gal-terminated saccharides that are abundant in human upper respiratory epithelium. There was substitution at position 627 (E→K) in the PB2 protein, which enhances the ability of the virus infection on the human. [Conclusion] This study provided a direct evidence for human as “mix vessel” of influenza virus, and the reassortment of seasonal influenza viruses and avian H7N9 viruses should be concerned.