Real-time reverse transcriptase PCR (rRT-PCR) assays have greatly contributed to the detection, control, and prevention of the pandemic influenza A/H1N1 2009 virus. To improve the rRT-PCR assays for detection of pandemic influenza A/H1N1 2009 virus, we evaluated the sensitivity, specificity, and performance of 12 rRT-PCR primer-probe sets [SW (a) to SW (l)] using a panel of virus strains and clinical specimens. These primer-probe sets were derived from published work and designed for detecting the hemagglutinin (HA) or the neuraminidase (NA) gene of the pandemic influenza A/H1N1 2009 virus. A primer-probe set, SW (CDC), developed by the Centers for Disease Control and Prevention (U.S. CDC) to target the HA gene of pandemic influenza A/H1N1 2009 virus, was used as a referee method. Our results demonstrated that although all primer-probe sets in this study had as high as 98.4 to 100% in silico coverage, some of the primer-probe sets had better specificity, sensitivity, and amplification efficiency than others. Two primer-probe sets, SW (h) and SW (l), which target the NA gene of pandemic influenza A/H1N1 2009 virus, were highly sensitive (10(4) copies/reaction), had high detection rates (56/60, P = 0.134, and 59/60, P = 1.000), and showed ideal specificity compared with SW (CDC). In addition, a cocktail of primer-probe sets targeted to the HA and NA genes displayed higher detection sensitivity than primer-probe sets targeting HA or NA alone, indicating that for practical applications, a combination of primer-probes targeting HA and NA genes is the best option for the detection of pandemic influenza A/H1N1 2009 virus.