This study was designed to estimate the prevalence of Chlamydia infection in women attending Obstetrics and Gynaecology clinics in Tehran, during May 2003 to October 2003. Women attending Obstetrics and Gynaecology clinics aged 15-42 were recruited by Sequential Random Sampling. Those who had not passed urine in the last hour were eligible. Informed consent was obtained and a questionnaire completed after being interviewed by a midwife. First void urine was collected and after DNA extraction from urine specimen, PCR tests were performed; urine DNA samples were tested by strand displacement amplification (SDA) for Chlamydia confirmation. 12.6% (133/1052) tested positive for Chlamydia by PCR. Of these PCR positive samples, 86 were available for re-testing by SDA and 67 were positive giving a correlation between the tests of 78%. This gave an overall true prevalence of 6.4% which is however, underestimated. No statistical differences were seen between patient age groups, details of personal and reproductive history and combined PCR and SDA positivity for C. trachomatis. A 12.6% prevalence of Chlamydia trachomatis was found by PCR testing which is cost effective to screen and treat. Despite limitations in re-testing PCR-positive samples by SDA, a 78% correlation between tests confirms a high prevalence of C. trachomatis. Non-invasive screening of women was therefore a success in this group of patients. As this was the first time that more sensitive molecular methods were used for detection of C. trachomatis, prevalence in such a big sample size, the results are considerable. However, we suggest further such testing.