An outbreak of human infections with a novel reassortant avian-origin H7N9 influenza virus occurred in several provinces of China in March 2013 (1). Unusually high proportion of severe cases and high rate of fatality has been observed in the patients with H7N9 virus infection (2). We reported recent emergence of NA R292K mutant strain and its association with the severe clinical outcome (3). Studies have shown that NA R292K mutation can cause the high level of Oseltamivir resistance in H7N9 avian influenza virus (4-5). In this study, we developed a single nucleotide polymorphism (SNP) TaqMan real-time RT-PCR assay to differentiate the NA 292K mutant and R292 wild-type (WT) of H7N9 virus in clinical samples. The low limits of detection (LODs) reached to 100 copies per reaction for 292K mutant and for R292 WT. It could differentiate 10% mutant strain from mixed viral population which is more sensitive than Sanger sequencing (with the detection threshold of 25% minor component in a mixed sample) (6). The assay is a sensitive, specific, fast and low-cost method for the rapid detection of the influenza A/H7N9 R292K oseltamivir resistance strain to monitor its circulation and transmission in the community and guide the antiviral treatment.