The prevalence of cytomegalovirus (CMV) infections ranges between 50% and 85% in adults in the United States, and its epidemiology varies in different regions of the world and between socioeconomic and age groups. In Portugal, no study has been carried out to date to determine the prevalence of CMV in the general population. Under the second National Serological Survey conducted in continental Portugal in 2001–2002, we estimated the prevalence of individuals with antibodies to CMV using indirect immunofluorescence to detect virus-specific IgG. The population sample included 2,143 individuals of both sexes and different ages from all 18 districts in Portugal. The national seroprevalence of CMV was determined as 77%. We analysed the proportion of CMV IgG by sex, age group and district of residence. This was the first nationally representative study of seroprevalence of CMV in Portugal. The results of the study indicate that CMV infection is highly prevalent in the population and occurs mainly in the first years of life.