Chlamydia trachomatis (CT) is the most common cause of bacterial sexually transmitted infection (STI) worldwide, but current data concerning the prevalence of CT among women in Iran is scarce. Data regarding the frequency of CT infection among Iranian women can help to justify the implementation of a national CT screening program that can reduce the high morbidity associated with sequelae of CT infections by treating infected women. Endocervical secretions from 123 married women (20–55 years) with cervicitis were tested by a PCR-EIA method using primers to amplify a CT-specific plasmid. The digoxigenin-labeled amplicon was measured by hybridization to a biotin-labeled probe and a strepavidin-coated plate, followed by an enzyme-linked colorimetric analysis. Overall frequency of CT infection among women was 17% (21/123). The range of CT frequency among various age groups was 12–25%. The 31–40-year-age group comprised the majority (49%) of CT positive samples, followed by 20–30 year group (33%). Although the 20-to-30-year-old women reported the highest frequency of STI history, they had the lowest relative frequency of CT infection (12%). There is a high frequency of CT infection among women with cervicitis in Tehran, Iran, thus indicating a necessity to implement a routine CT screening program in the major cities of Iran and possibly nationwide. Identification of CT-infected women may prevent its spread, and thereby reduce the high morbidity associated with CT infections among women in Iran.