Background: Nowadays, Chlamydia trachomatis is known as a causative agent of infertility. Because of, asymptomatic nature of infection, many may suffer from its lasting complications such as infertility. This study was performed in Tehran during April 2007 to April 2008 to compare the prevalence of Chlamydia trachomatis infection in fertile and infertile women using ELISA and PCR methods. Methods: Overall, 234 infertile and 223 pregnant women, as the fertile group, participated in this hospital-based case-control study. After completing an informed consent form and the questionnaire, first catch urine and blood sample were obtained for PCR and ELISA (IgG, IgM) tests, respectively. Logistic regression analysis was used to control possible confounding factors, and determine adjusted odds ratio of infertility due to the infection. Results: PCR results revealed that 29 (12.4%) of the infertile and 19 (8.5%) of the fertile women were positive for C. trachomatis infection (p=0.440). IgG was positive in 21 (9.0%) of the infertile and 11 (5.0%) in the fertile group (p=0.093). IgM assays identified that 2 (0.9%) of the infertile and 4 (1.8%) of the fertile women were positive for the micro-organism (p=0.375). Conclusion: We found no significant differences among fertile and infertile women for Chlamydia trachomatis infection. Nevertheless, molecular techniques which are more sensitive, more specific and non-invasive can be used to detect C. trachomatis infection.