De novo high-throughput pyrosequencing was used to detect and characterize 2009 pandemic influenza A (H1N1) virus directly in nasopharyngeal swabs in the context of the microbial community. Data were generated with a prior sequence independent amplification by 454 pyrosequencing on GS-FLX platform (Roche). Influenza A assembled reads allowed near full-length genome reconstruction with the simultaneous analysis of site-specific heterogeneity. The molecular approach applied proved to be a powerful tool to characterize the new pandemic H1N1 influenza virus in clinical samples. This approach could be of great value in identifying possibly new reassortants that may occur in the near future.