The single-nucleotide variation 823C to T (His275Tyr), responsible for oseltamivir drug resistance has been detected in some isolates of the influenza A/H1N1/2009 virus. Early detection of the presence of this oseltamivir-resistant strain allows prompt consideration of alternative treatment options. An isolated-probe-asymmetric amplification PCR (Roche LightCycler v2.0) and high-resolution melting (HRM) method using unlabeled probes and amplified products (Idaho LightScanner 32) was designed and optimized to detect and estimate the proportion of H275Y mutants in influenza A/H1N1/2009 virus samples. The lower limit of quantification within the linear range of PCR assay detection was 200 copies/reaction. The melting peaks of the H275Y-specific unlabeled probe for the wild-type A/H1N1/2009 and H275Y mutant viruses were clearly distinguishable at 65.5°C and 69.0°C, respectively, at various ratios of wild-type/mutant virus population standards. The 95% detection limit for the 10% mutant sample pool was 1,200 copies/reaction (95% confidence interval, 669.7 to 3,032.6 copies/reaction). This HRM assay was tested with 116 archived clinical specimens. The quantitative HRM results obtained with samples containing mixed mutant-wild-type virus populations, at threshold cycle (C(T)) values of <29, compared well to those obtained with a pyrosequencing method performed by an independent laboratory. The quantitative feature of this assay allows the proportions of mutant and wild-type viral populations to be determined, which may assist in the conventional clinical management of infected patients and potentially more preemptive clinical management. This validated quantitative HRM method, with its low running cost, is well positioned as a rapid, high-throughput screening tool for oseltamivir resistance mutations in influenza A/H1N1/2009 virus-infected patients, with the potential to be adapted to other influenza virus species.