In the beginning of 2007-2008 Northern Hemisphere influenza season, the frequency of influenza A(H1N1) viruses bearing a previously defined oseltamivir resistance conferring amino acid change of Histidine to Tyrosine at position 274 (H274Y) of the neuraminidase (NA) increased dramatically. In order to rapidly detect such resistant viruses, an RT-PCR/restriction fragment length polymorphism (RT-PCR/RFLP) assay targeting amino acid 274 of the N1 NA molecule was developed to investigate the presence or absence of the H274Y mutation. The reverse primer was engineered to produce a BspHI site in the amplicon for oseltamivir-sensitive viruses with Histidine at position 274 (274H). A total of 50 influenza A(H1N1) specimens including 30 oseltamivir-sensitive and 20 oseltamivir-resistant ones submitted to the Centers for Disease Control and Prevention (CDC) during the 2007-2008 influenza season were successfully characterized by this assay. The assay was specific for grown A(H1N1) viruses and original clinical specimens, with a lower limit of detection of approximately 10 RNA transcript copies per reaction. Our RT-PCR/RFLP assay provides a simple, rapid and sensitive tool to monitor the emergence and spread of H274Y oseltamivir-resistant influenza A(H1N1) viruses.