Abstract: Objective To investigate the difference of pandemic and post-pandemic influenza A (H1N1pdm09) viruses among epidemiological distribution, evolution, epitope and drug-resistant substitutions. Methods The comparative analysis of pandemic and post-pandemic viruses on phylogenetic trees, evolutionary rate and amino acid mutations of the hemagglutinin, and neuraminidase genes in Hubei from 2009 to 2014 were conducted. Results Positive rate of nucleic acid testing for pandemic viruses was higher than post-pandemic viruses, but evolutionary rate was slower, and evolutionary rate of neuraminidase gene was faster than hemagglutinin gene in the both periods. Amino acid substitutions of hemagglutinin epitopes, such as K163Q, A186T, S185T and S203T, had different distributional and positional preference between pandemic and post-pandemic viruses. Drug-resistance site H275Y substitution was found on three post-pandemic viruses, but little substitution had been reported on pandemic viruses before. Conclusions The evolutionary rate, epitope mutation and drug-resistant substitutions of post-pandemic influenza A (H1N1pdm09) viruses are increasing. The situation of prevention and control is grim. Surveillance of influenza A (H1N1pdm09) viruses should be strengthened especially for genetic feature and analysis of drug resistance.