Knowledge of seroprevalence rates against 2009 pandemic H1N1 virus will assist vaccination recommendations and the preparation of the health-care system during subsequent years. This study was conducted in Greece during June-August 2010 to estimate the seroprevalence rate against pandemic H1N1 virus. Persons presenting in 29 health-care facilities across the country were studied. Seroprevalence was estimated employing a virus-free ELISA that specifically recognizes 2009 H1N1 virus antibodies in human sera. Sera collected from 2005 to April 2009 were also used to estimate pre-pandemic seroprevalence rates. A total of 954 persons were studied. The overall seroprevalence rate was 28.5% (95% confidence interval=25.6-31.3%). Age-specific rates were 34.2% in persons 0-4 years, 36.3% in persons 5-19 years, 25.0% in persons 20-39 years, 23.4% in persons 40-59 years, and 31.8% in persons ≥ 60 years. The highest rates were recorded in the Regions of Ionian Islands (67%) and Epirus (42.9%), while the lowest (8.4%) in the Region of Thessaly. Age-specific attack rates of infection during 2009-2010 were 28.8% in persons 0-4 years, 32.5% in persons 5-19 years, 14.3% in persons 20-39 years, 19.1% in persons 40-59 years, and 14.4% in persons ≥ 60 years. Multivariate analysis revealed that Region of residence and caring for children <5 years were associated with increased risk for seropositivity. Urbanity, personal and family characteristics, working in a health-care facility or in a school, history of pandemic H1N1 vaccination or history of influenza-like illness during 2009-2010 were not associated with increased risk for seropositivity.