Two cases of febrile respiratory illness associated with untypeable influenza A virus were identified in Southern California in March 2009. One was initially detected as influenza virus using an experimental diagnostic device in a clinical trial, while the other was detected at a local reference lab using a diagnostic PCR assay. In both cases, analyses yielded negative results for strain-specific tests targeting circulating strains of influenza A virus (seasonal H1 and H3). These two samples became the first reported cases of the pandemic 2009/H1N1 influenza virus. The first reportable characterization was made from the second collected specimen on 15 April 2009 at the Centers for Disease Control and Prevention central lab using traditional culture and sequencing methods. The novel nature of the strain and its apparent zoonotic origins were initially characterized using the first collected specimen at the Naval Health Research Center in San Diego, CA, on 13 April using an experimental molecular analysis tool, PCR electro-spray ionization-mass spectrometry (PCR/ESI-MS), designed to amplify PCR products from any strain of influenza virus and to generate informative (phylogenetic) strain identifications through mass spectrometry of PCR amplicons. The ability of this high-throughput tool to correctly identify both well-characterized and novel influenza strains offers the possibility to integrate surveillance for emerging strains with on-site rapid diagnosis used for patient management, shortening the times between the emergence of new strains, their detection and identification, and appropriate public health response activities. Here we describe the initial characterization of the pandemic 2009/H1N1 influenza strain and discuss the possible roles of diagnostic tools with discovery potential.