Accurate and rapid diagnosis of Pandemic Influenza A/H1N1 2009 virus (H1N1 2009) infection is important for the prevention and control of influenza epidemics and the timely initiation of antiviral treatment. This study was conducted to evaluate the performance of several diagnostic tools for the detection of H1N1 2009. Flocked nasopharyngeal swabs were collected from 254 outpatients of suspected H1N1 2009 during October 2009. This study analyzed the performances of the RealTime Ready Inf A/H1N1 Detection Set (Roche), Influenza A (H1N1) Real-Time Detection Kit (Bionote), Seeplex Influenza A/B OneStep Typing Set [Seeplex Reverse Transcriptase PCR (RT-PCR)], BinaxNow Influenza A & B Test Kit [Binax Rapid Antigen Test (RAT)], and SD BIOLINE Influenza Ag kit (SD RAT). Roche and Bionote real-time RT-PCR showed identical results for the H1N1 2009 hemagglutinin gene. Compared with real-time RT-PCR, the sensitivities and specificities were 83.7% and 100% for Seeplex RT-PCR, 64.5% and 94.7% for Binax RAT, and 69.5% and 100% for SD RAT. The sensitivities of Seeplex RT-PCR, Binax RAT, and SD RAT in patients aged over 21 years were 73.7%, 47.4%, and 57.9%, respectively. The sensitivities of Seeplex RT-PCR, Binax RAT, and SD RAT on the day of initial symptoms were mostly lower (68.8%, 56.3%, and 31.3%, respectively). In conclusion, multiplex RT-PCR and RAT for the detection of H1N1 2009 were significantly less sensitive than real-time RT-PCR. Moreover, a negative RAT may require more sensitive confirmatory assays, because it cannot be ruled out from influenza infection.