A C-to-T transition mutation in the neuraminidase gene from seasonal A/H1N1 causes a His-to-Tyr mutation at amino acid position 275 (H274Y, universal N2 numbering), conferring resistance against oseltamivir (Tamiflu). This mutation was first detected in clinical samples in Europe during the 2007-2008 influenza season. Viruses with this mutation reached a prevalence of ∼11% by the end of the season in North American isolates tested by the CDC. We developed a highly sensitive and specific quantitative real-time reverse transcriptase PCR assay to detect the H274Y mutation. This assay utilizes a 5'-methyl-isocytosine (isoC) residue and fluorescent reporters on genotype-specific primers. During PCR, a quencher coupled to isoguanine (isoG) is site-specifically incorporated complementary to the isoC/dye, resulting in loss of fluorescence. Optimization of primers and assay conditions produced a limit of detection of 100 gene copies per reaction for both wild-type and H274Y genotypes. In samples with mixed populations, it can reliably detect as little as a 1% wild-type or 0.1% H274Y component. This high sensitivity makes the assay usable on samples with viral loads too low for dideoxy or pyrosequencing analysis. Additionally, the assay distinguishes seasonal A/H1N1 from A/H3N2, influenza B, or 2009 pandemic A/H1N1, making it useful for influenza virus subtyping as well as for drug resistance detection. We probed seasonal A/H1N1 samples from the 2005-2006, 2006-2007, and 2007-2008 influenza seasons. Data from the new assay closely matched available drug resistance genotype data previously determined by dideoxy sequencing. The H274Y mutation was only found in samples from the 2007-2008 season.