Chlamydia pneumoniae (CPN) causes pneumonia in humans, and has emerged as an important respiratory pathogen. There are also established links between CPN infection and coronary artery disease. Traditional culture methods for CPN detection can be time consuming and difficult. There are a variety of molecular-based amplification methods for CPN detection. These methods are more sensitive than culture, but have the disadvantage of being inconsistent and non-comparable across studies. In this paper, we describe the adaptation of the existing primer set CPN 90/CPN91 for use in a real-time PCR assay using the Roche Lightcycler and a Taqman probe. This assay had an analytical sensitivity of between 4 and 0.4 infection-forming units (IFUs)/PCR reaction. A total of 355 samples were tested for validation of the assay. Tested samples included two standardized panels of blinded samples from culture (N = 70), archived specimens consisting of a CPN dilution series, CPN-spiked porcine aortal tissue and endarterectomy specimens (N = 87). The third group consisted of prospectively collected PBMCs from clinical samples (N = 198). Results were compared to nested PCR, which targets the ompA gene of CPN; TETR PCR, which targets the 16S rRNA gene of CPN; or the known result for the sample. Overall, the assay had a sensitivity of 88.5% (69 of 78) and a specificity of 99.3% (275 of 277). This method should prove useful for accurate, high throughput detection of CPN.