Previously published PCR-based diagnostic tests for melioidosis were evaluated for clinical usefulness. A Burkholderia pseudomallei 16S rRNA-derived primer set had a sensitivity approaching 100% for clinical samples from 22 culture-confirmed cases of melioidosis and enabled diagnosis of 3 culture-negative cases. However, samples from 10 of 30 inpatients from Royal Darwin Hospital with other diagnoses were positive by PCR, giving a specificity of 67% and a positive predictive value of only 70%. Although there are a number of intriguing possible explanations for our results, concerns of inappropriate therapy resulting from a positive result by PCR have led us to forgo the advantage of rapid PCR diagnosis for melioidosis until a better system is validated.