Oseltamivir has been used widely for prophylaxis or treatment during outbreaks of the pandemic influenza virus (H1N1) in several countries. The aim of this study was to develop a real-time RT-PCR (reverse transcription-polymerase chain reaction) to be applied for detection and monitoring of the oseltamivir resistant strains of this virus during three outbreaks (May, 2009 to October, 2010) in Thailand. The real-time RT-PCR assay for detecting H275Y proved highly specific for the pandemic influenza virus (H1N1) as no cross-amplification was detected with other respiratory viruses or human total RNA. The assay was also highly sensitive with a detection limit as low as 100 copies/μL for both wild-type and resistant strains. The performance of the assay was evaluated in terms of amplification efficiency (100%). The results obtained by real-time RT-PCR were in complete agreement with direct nucleotide sequencing. However, real-time RT-PCR provided more detail on the relative quantities of ratios between resistant and sensitive strains in each individual. The results revealed that four of 1,288 (0.31%) patients were infected with the oseltamivir resistant strain. The number of patients infected by resistant strains was higher during the third (0.61%) and second (0.24%) waves than during the first (0%) outbreak. In conclusion, the real-time RT-PCR assay for H275Y detection is advantageous because it is specific, sensitive, and provides quantitative data. And it would be useful for large-scale testing and monitoring of oseltamivir resistant strains of the pandemic influenza A virus (H1N1).