The single nucleotide variant (SNV), 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 (IPAA-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 detection was 200 copies/reaction. The melting peaks of the H275Y-specific unlabeled probe for 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 1200 copies/reaction (95% CI: 669.7-3032.6 copies/reaction). This HRM assay was tested on 116 archived clinical specimens. The quantitative HRM results of samples containing mixed mutant-wild-type virus population, at Ct value <29, compared well against a pyrosequencing method performed by an independent laboratory. The quantitative feature of this assay allows the proportion of mutant:wild-type viral populations to be determined, which may assist in the conventional and potentially a more pre-emptive clinical management of such infected patients. 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 infected patients, with the potential to be adapted for other influenza species.