Introduction: Chlamydiosis is a worldwide zoonotic disease caused by different microorganisms in the order Chlamydiales. The aim of this study was to detect and determine the prevalence of Chlamydia infection in pet and zoo reptiles in Tehran, Iran. Materials and Methods: In a period of 10 months from April 2015 to February 2016, swab samples were collected from cloaca and conjunctiva of 130 pet or zoo reptiles (18 snakes, 81 turtles, and 31 iguanas). A Real Time-PCR assay targeting 23s rRNA of chlamydial organisms was performed to detect chlamydial infection in clinical specimens. Results: No positive sample could be detected in the investigated clinical specimens in the present study. Conclusion: Regarding the negative results which were achieved in this study, reptiles could not be important hosts of chlamydial organisms at least in the region of the present study, Iran, Tehran. Despite the present findings in reptiles, pet and aviary birds were previously shown to be remarkable host of Chlamydia spp. in Iran. Further studies particularly serologic surveys and other PCR methods are needed to thoroughly evaluate significance of the chlamydial infection in reptiles. A rapid, accurate and cost-effective method was applied for Chlamydiaceae spp detection and discrimination of the most significant Chlamydia spp., causing disease complications in reptiles. The results indicated low zoonotic risk of Chlamydia spp in Iranian reptiles.