Since April 2009, the outbreak of human pandemic influenza A (H1N1) virus (pH1N1) infection has spread from North America to other parts of the world, and currently, pH1N1 is the predominant circulating strain of influenza viruses. Our objectives were to perform a serological survey of medical personnel at the Chumphae Hospital in Thailand and to investigate the prevalence of pH1N1 in randomly selected patients diagnosed with respiratory tract disease. Prevalence of pH1N1 in the patients was determined by performing real-time reverse transcription-polymerase chain reaction. The study was carried out between July 2009 and November 2010. Seroprevalence of hemaglutination inhibition (HI) titers among medical personnel was established in three cross-sectional studies at the end of each wave of the pandemic by performing HI assay to detect antibodies against pH1N1. Infection by the pH1N1 peaked between July and October 2009; the second wave was from January to March 2010 and the third wave from June to November 2010. The HI titers after the first, second, and third waves were 48.2%, 22.4%, and 25.7%, respectively. After the second and third waves, 52.1% and 45.3% of the medical personnel who had received pH1N1 vaccination had HI titers ≥ 40. These findings show that seasonal influenza strain in Chumphae and the predominant influenza strain from each wave was pH1N1. HI assay results also represent the severity of the attack rate in each wave.