Diagnosis of Q fever has largely been dependent upon serology, which may lead to delayed diagnosis as seroconversion can take weeks to develop. During a recent Q fever outbreak (27 patients) in rural South Australia, we compared the diagnostic rate of serology with two separate real-time PCRs, the 27kDa outer membrane protein and the insertion sequence. PCR was positive (on either or both PCR assays) in sera of 67% of the patients. Median time required for making serological diagnosis was 17 days, compared with 4 days by PCR. Q fever PCR is an effective tool in the diagnosis of acute Q fever infection.