Five epidemic waves of human infection with influenza A (H7N9) virus have emerged in China since spring 2013. We previously described the epidemiological characterization of the fifth wave in Jiangsu province. In this study, 41 H7N9 viruses from patients and live-poultry markets were isolated and sequenced to further elucidate the genetic features of viruses of the fifth wave in Jiangsu province. Phylogenetic analysis revealed substantial genetic diversity in the internal genes, and 18 genotypes were identified from the 41 H7N9 virus strains. Furthermore, our data revealed that 41 isolates from Jiangsu contained the G186V and Q226L/I mutations in their haemagglutinin (HA) protein, which may increase the ability of these viruses to bind the human receptor. Four basic amino acid insertions were not observed in the HA cleavage sites of 167 H7N9 viruses from Jiangsu, which revealed that highly pathogenic avian influenza (HPAI) H7N9 viruses did not spread to Jiangsu province in the fifth wave. These findings revealed that multiple genotypes of H7N9 viruses co-circulated in the fifth wave in Jiangsu province, which indicated that the viruses have undergone ongoing evolution with genetic mutation and reassortment. Our study highlights the need to constantly monitor the evolution of H7N9 viruses and reinforce systematic influenza surveillance of humans, birds, and pigs in China.