Background:Chlamydia trachomatis (C. trachomatis) is the most common bacterial sexually transmitted infection (STI). Although most genital C. trachomatis infections remain asymptomatic but infection with these bacteria is the leading cause of complications, such as pelvic inflammatory disease (PID), tubal factor infertility and abortion. Objectives: The objective of this study was to estimate the frequency of C. trachomatis infection among symptomatic and asymptomatic women, by a polymerase chain reaction (PCR) based assay. Patients and Methods: This was a cross-sectional study conducted over the period from May 2012 to February 2013. A total of 240 nonduplicate endocervical samples were taken from married women; endocervical swabs were collected from women referred to Qazvin Kowsar Gynecology Hospital by a training midwife. The specimens were tested for C. trachomatis by a PCR-based assay for the pha gene. Results: Out of the 240 female participants, 184 (76.7%) were symptomatic and 56 (23.3%) were asymptomatic cases. The mean age of cases was 37.1 ± 0.9 years. Twenty (8.3%) of the 240 samples were diagnosed as Chlamydia positive according to PCR results. The prevalence of asymptomatic C. trachomatis infections was six (10.7%), while there were 14 (7.6%) in symptomatic cases. Although positive PCR results have shown in women with vaginosis (7.1%), abortion (5.1%), premature birth and low birth weight below 2.5 kg (7.7%) but the chi-square test did not indicate a significant relationship between positive PCR test results and these symptoms. Conclusions:The results of this study showed that there was a high prevalence of C. trachomatis infection among both symptomatic and asymptomatic women. Therefore, a screening test for C. trachomatis infection is recommended for all women who refer to the genitourinary medicine clinic. Screening programs are important for cost effectiveness calculations of C. trachomatis infections especially in asymptomatic cases.