Background: Chlamydia trachomatis is one of the most common diseases as sexually transferred in world. According to the World Health Organization statistics, approximately 92 million new Chlamydia trachomatis infection occur in the world. Chlamydia trachomatis (CT) is the cause of tubal obstruction, ectopic pregnancy and infertility in women. The aim of this study is prevalence evaluation of Chlamydia Trachomatis by PCR in female infertile referred to Montasarieh study center of infertility in Mashhad. Materials and Methods: The cervical swab specimens were collected from 100 infertile (as case) and 30 fertile (as control group) women attending to the infertility center of Mash-had Medical University. DNA extraction was performed from clinical specimens using DNA extraction kit. In this study, in addition to PCR reaction by commercial kit, PCR test was performed using specific primers and probe for CTCP gene. Results: The results of PCR reaction using the kit was match with PCR test and showed that the prevalence of Chlamydia trachomatis is 21% in infertile women and 3.3% in normal fertile women that was statistically significant (p=0.024). Conclusion: Considering the high sensitivity of PCR method for diagnosis of Chlamydia trachomatis infection, this method can be useful for routine screening.