We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.