BACKGROUND: Real-time reverse transcriptase PCR (rRT-PCR) is widely used to detect novel influenza A/H1N1. We had observed several cases with positive result for influenza A and negative result for novel influenza A/H1N1 during a novel influenza A/H1N1 outbreak. The causes of those results were investigated in this study. METHODS: A total of 913 cases tested with rRT-PCR for novel influenza A/H1N1 (Real-time Ready Influenza A/H1N1 Detection Set, Roche Diagnostics, Germany) during 25 August 2009 to 8 September 2009 was enrolled in this study. Cases showing positive result for influenza A (M gene) and negative result for novel influenza A/H1N1 (H1 gene) were tested with multiplex RT-PCR for seasonal influenza and novel influenza A/H1N1 (Seeplex FluA ACE Subtyping kit, Seegene, Korea), and the amplicons were directly sequenced. RESULTS: One hundred and eleven cases (12.2%) were positive for novel influenza A/H1N1. Twenty-seven cases (3.0%) were positive for influenza A, but negative for novel influenza A/H1N1. Subtypes of influenza A were determined in 25 cases by multiplex RT-PCR and nucleotides sequencing. One novel influenza A/H1N1, six seasonal influenza A/H1N1, three seasonal influenza A/H3N2, and 15 influenza A/H9N2 were detected. CONCLUSIONS: Subtypes of influenza A were determined in most cases with positive result for influenza A and negative result for novel influenza A/H1N1. Several cases with seasonal influenza A were detected. Even if a nonepidemic period of seasonal influenza, tests for seasonal influenza A can help in the differential diagnosis of influenza.