During the 2009 novel influenza (H1N1) pandemic, the sensitivity of direct immunofluorescence assay (DFA) for H1N1 infection was 62% (266/429) of that of the polymerase chain reaction (PCR) test. The sensitivity of the DFA differed significantly with the age of patients: the sensitivity was the highest (71.8%) for patients aged <10 years and the lowest for patients aged ≥30 years. The sensitivity of DFA in patients aged ≥30 years was 40.7%. Furthermore, the sensitivity (67.3%, 171/254) of DFA was higher for patients who had a high temperature at admission. An increase in the incidence of H1N1 infection did not influence the sensitivity of DFA (62.1% vs. 62%; p=0.984) test, but resulted in a decrease in the negative predictive value, from 92.4% (700/757) to 69.6% (247/355). PCR may be useful as the initial test for diagnosing H1N1 infection in patients aged ≥30 years with a normal temperature at presentation.