There were three epidemic waves of human infection with avian influenza A (H7N9) virus in 2013-2014. While many analyses of the genomic origin, evolution, and molecular characteristics of the influenza A (H7N9) virus have been performed using sequences from the first epidemic wave, genomic characterization of the virus from the second epidemic wave has been comparatively less reported. In this study, an in-depth analysis was performed with respect to the genomic characteristics of 11 H7N9 virus strains isolated from confirmed cases and four H7N9 virus strains isolated from environmental samples in Shenzhen during the second epidemic wave. Phylogenetic analysis demonstrated that six internal segments of the influenza A (H7N9) virus isolated from confirmed cases and environmental samples in Shenzhen were clustered into two different clades and that the origin of the influenza A (H7N9) virus isolated from confirmed cases in Shenzhen was different from that of viruses isolated during the first wave. In addition, H9N2 viruses, which were prevalent in southern China, played an important role in the reassortment of the influenza A (H7N9) virus isolated in Shenzhen. HA-R47K and -T122A, PB2-V139I, PB1-I397M, and NS1-T216P were the signature amino acids of the influenza A (H7N9) virus isolated from confirmed cases in Shenzhen. We found that the HA, NA, M, and PA genes of the A(H7N9) viruses underwent positive selection in the human population. Therefore, enhanced surveillance should be carried out to determine the origin and mode of transmission of the novel influenza A (H7N9) virus and to facilitate the formulation of effective policies for prevention and containment of a human infection epidemics.