The epidemiology of Q-fever in Iran is essentially unknown. This study was conducted to determine the prevalence rate of Coxiella burnetii in bulk milk samples from dairy bovine, ovine, and caprine herds in Chaharmahal va Bakhtiari province, Iran. In this study, 376 bulk milk samples from 79 dairy bovine, ovine, and caprine herds were tested for C. burnetii using a nested PCR assay. The animals whose milk samples collected for this study were clinically healthy. In total, 13 of 210 (6.2%) bovine milk samples were positive; the positive samples originated from 5 of 28 (17.9%) commercial dairy herds. All 110 ovine bulk milk samples from 31 sheep breeding farms were negative and only 1 of 56 (1.8%) caprine bulk milk samples from 20 goat breeding farms was positive for C. burnetii. Although no extensive prevalence study was undertaken, the results of this study indicate that clinically healthy cattle are important sources of C. burnetii infection in Iran. To our knowledge, this study is the first report of direct identification of C. burnetii by PCR in bulk milk samples from dairy bovine and caprine herds in Iran. Further intensive prevalence studies on Coxiella infection among farmers, milk-processing workers, veterinarians, and slaughterhouse workers and on possible dangers of dairy products will be needed to elucidate the epidemiology of Q fever in Iran.