Chlamydia trachomatis is a common cause of sexually transmitted disease which can cause severe consequences. Effective prevention requires knowledge of prevalence of infection in order to target interventions in a cost-effective manner. To determine the prevalence of chlamydial infection in Mashhad, northeastern Islamic Republic of Iran, this study was performed among male patients with urethritis. Urethral discharge was collected from 150 patients. Cell culture was established for diagnosis of Chlamydia in genital specimens. Cell culture showed that 9.3% of patients in this study were infected with Chlamydia. This study provides strong evidence that prevalence of Chlamydia in our region is quite high, which necessitates screening and treatment for the infection.