Despite the widespread distribution of Q fever, the prevalence in humans is not accurately known, because many infected people seroconvert without symptoms or with a mild febrile disease. The aim of this study was to determine the seroprevalence of Q fever in different regions of Croatia. During a 2-year period (2008-2010), serum samples from 552 febrile patients with prolonged cough aged 1-88 were tested for the presence of Coxiella burnetii antibodies by using indirect immunofluorescent assay. Sera from 27.5% patients showed IgG antibodies. Serological evidence of C. burnetii infection was found in patients from all parts of Croatia. Seroprevalence rates significantly differed among regions from 21.5% to 41.2% (p=0.001). Men were more often seropositive (31.6%) than women (22.2%; p=0.016). According to age, a progressive increase in the IgG seropositivity rates was observed as ranging from 6.7% in children less than 10 years of age to 39.2% in patients aged 40-49 (p=0.001). Above the age of 50, the IgG seroprevalence remained stable. Patients from rural areas were more often seropositive than patients from urban areas (40.8% vs. 19%), p<0.001). Acute Q fever was confirmed in 5.8% of patients. Cases occurred throughout the year. A majority of cases were reported during summer months.