Patients with the underlying valvular heart disease are at the high risk of developing sub-acute or chronic endocarditis secondary to Coxiella burnetii. Q fever endocarditis is the most common manifestation along with persistent the infection. There is some serologic and molecular evidence of C. burnetii infection in humans and livestock in Iran. As it is possible to observe chronic Q fever in Iran, it seems necessary to study the prevalence of Q fever endocarditis in this country. In the present study, Infective Endocarditis (IE) patients (possible or definite based on Duke Criteria) hospitalized in Rajaie Cardiovascular Medical and Research Center were enrolled from August 2016 to September 2018. Culture-negative endocarditis patients were evaluated by Raoult criteria for diagnosis Q fever endocarditis. The serological results for brucellosis were negative for all subjects. All blood and tissue samples including valve samples were tested for C. burnetii infection using serology and Polymerase Chain Reaction (PCR). In this study, 126 patients who were admitted to the hospital were enrolled; of which 52 subjects were culture-negative IE. Among the participants, 16 patients (30.77%) were diagnosed with Q fever IE and underwent medical treatment. The mean age of patients was 46.6 years ranging from 23 to 69 years and 75% of them were male. Considering the high prevalence of Q fever IE, evaluation of the patients with culture-negative IE for C. burnetii infections was highly recommended.