ABSTRACT Neuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.