One of the most common bacterial infections that causes ophthalmia neonatorum is Chlamydia trachomatis, (C. trachomatis). Very few studies have been performed in Iran using both cell culture and polymerase chain reaction (PCR) methods to determine the prevalence of C. trachomatis as an etiological agent of ophthalmia neonatorum. This study aimed to evaluate the prevalence of neonatal chlamydial conjunctivitis (NCC) as diagnosed by both methods in two hospitals in Tehran, Iran. From March 2008 to May 2009, out of 2253 neonates, 241 (10.7%) with clinical findings of conjunctivitis were included in this study. A total of 241 conjunctival swabs were tested by cell culture (as the gold standard test), PCR, and Giemsa staining. Cell cultures were positive for C. trachomatis in 31 (12.9%) neonates, C. trachomatis was positive in 40 (16.6%) neonates by PCR and 18 (7.5%) by Giemsa staining. The sensitivity of PCR was 100%, whereas Giemsa staining sensitivity was 558.1%. High sensitivity (100%) and specificity (95.7%) of PCR as compared to culture makes it a proper diagnostic method for the detection of C. trachomatis.