<p>Background: <em>Chlamydia trachomatis</em> is the etiology of most of sexually transmitted diseases. Colonization of <em>C. trachomatis</em> in the genital tract during early gestation has been associated with preterm birth, and preterm premature rupture of the membranes. The role of <em>C. trachomatis</em> on spontaneous abortion has not yet been proved completely. The aim of this study was to evaluate the frequency of <em>C. trachomatis</em> infection among pregnant women and its association with spontaneous abortion.<br /> Methods: This case-control study was conducted from August 2012 until January 2013. Totally, 218 women were included; 109 women with spontaneous abortion with gestation age between 10-20 weeks (cases), and 109 women with normal pregnancy with gestation age between 20-30 weeks (controls) in Sanandaj, Iran. DNA was extracted from endocervical swabs and a PCR test was conducted for detection of <em>C. trachomatis</em> infection in women using specific primers. Independent T-test and Chi-square were used for comparison of quantitative and qualitative variables, respectively, and p&lt;0.05 was considered significant.<br /> Results: The total prevalence of <em>C. trachomatis</em> infection was 38(17.43%) in endocervical swabs of women. However, the number of cases with <em>C. trachomatis</em> infections was 25 out of 109(22.9%) in the case group and 13 out of 109(11.9%) in control group, respectively. Association between chlamydia infection and spontaneous abortion was statistically significant (OR=2.198, CI 95%: 1.058-4.56).<br /> Conclusion: Our study showed that <em>C. trachomatis</em> infection was associated with spontaneous abortion. Thus, screening and treatment of pregnant women may prevent this adverse pregnancy outcome.</p>