Background. Signature amino acids of H7N9 influenza A virus play critical roles in human adaption and pathogenesis, but their dynamic variation is unknown during disease development. Methods. We sequentially collected respiratory samples from H7N9 patients at different timepoints and applied next-generation sequencing (NGS) to the whole genome of the H7N9 virus to investigate the variation at signature sites. Results. A total of 11 patients were involved, from whom 29 samples were successfully sequenced, including samples from multiple timepoints in 9 patients. Neuraminidase (NA) R292K, basic polymerase 2 (PB2) E627K, and D701N were the 3 most dynamic mutations. The oseltamivir resistance-related NA R292K mutation was present in 9 samples from 5 patients, including 1 sample obtained before antiviral therapy. In all patients with the NA 292K mutation, the oseltamivir-sensitive 292R genotype persisted and was not eliminated by antiviral treatment. The PB2 E627K substitution was present in 18 samples from 8 patients, among which 12 samples demonstrated a mixture of E/K and the 627K frequency exhibited dynamic variation. Dual D701N and E627K mutations emerged but failed to achieve predominance in any of the samples. Conclusions. Signature amino acids in PB2 and NA demonstrated high polymorphism and dynamic variation within individual patients during H7N9 virus infection.