There are limited data on the aetiology of acute undifferentiated febrile illnesses (AUFIs) in Iran. Moreover, Coxiella burnetii has not previously been detected in clinical samples in this country. Previous studies have highlighted the importance of considering C. burnetii as a cause of AUFI. In this retrospective study, in 92 cases of AUFI where Q fever was suspected, C. burnetii DNA was detected in seven samples (7.36%). This is the first molecular confirmation of C. burnetii from clinical samples from Iran.