Abstract: Here, we presented a Chlamydiaceae-specific 23S rRNA-based real-time PCR assay for simultaneous detection and quantitation of four members of Chlamydiaceae family, C. trachomatis, C. psittaci, C. pneumoniae and C. pecorum, using SYBR Green and Lightcycler^TM. The assay was characterized using plasmid constructs of the bacteria and verified on standard strains of all four species of the Chlamydiaceae and a large cohort of clinical samples collected from human and animals by comparison with fluorescence immunohistochemistry method. The results showed that the present real-time PCR assay was of high specificity and sensitivity. It was capable of detecting as few as 250 fg of chlamydial DNA (equivalent to 10^-1 IFU) and was applicable to both liquid cultures and clinical samples. This assay may therefore offer a rapid, economic and reliable method for screening of the chlamydiaceae pathogens.