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Background Chlamydia trachomatis (CT) infection is the most common sexually transmitted disease in the world that can persist and also ascend in the genital tract. This intracellular and silent infection is related to some adverse pregnancy outcomes, such as miscarriage. The aims of this study were to explore the best CT screening tests using blood and vaginal samples and to investigate the correlation between CT infection and the incidence of miscarriage. Materials and Methods This case-control study was done in October 2013 through June 2014, using purposive sampling from 157 female participants with or without a history of miscarriage. The samples were taken after each participant had signed a letter of consent and had completed a questionnaire. To achieve the objectives of this study, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests were performed on vaginal swabs and blood samples, respectively. Results PCR results showed a significantly higher CT infection rate in the miscarriage group compared to the control group (11.3 vs. 0%, P=0.007). Anti-CT IgG and IgA antibodies were found in 4.2 and 2.1% of cases in the miscarriage group, and in 1.7 and 6.7% of cases in the control group, respectively (P > 0.05). Despite lower humoral responses in this study, positive samples were detected only by one of the following techniques; PCR, ELISA IgA and ELISA IgG. It also should be noted that PCR worked best in terms of detection. Conclusion Based on the obtained data, there is a strong association between molecular evidence of CT infection and miscarriage. A higher rate of CT detection in molecular tests compared to serological assays suggests that PCR could be used as the first-choice assay for detection of C. trachomatis. However, the importance of serological tests in detecting potential past CT infection or upper genital infection not amenable to sampling is undeniable.