In India, given the scarce availability of sensitive and specific methods, Chlamydia trachomatis genital infections may lead to severe clinical complications when left undiagnosed or underdiagnosed. The present study was conducted to evaluate the diagnostic efficiency and feasibility of polymerase chain reaction (PCR) assays using genital and urine specimens from men and women in India. Genital swabs and urine specimens collected from 143 patients attending the sexually transmitted disease (STD) clinic, Government General Hospital, Chennai, were tested by culture and a plasmid based PCR. Culture was positive in 27 (18.9%) patients. PCR gave positive results for 46 (32.2%) cases using genital specimens, and the positivity rate in urine was 25.2%. Once the discordant results between culture and PCR had been resolved by using a major outer membrane protein PCR, the overall sensitivity, specificity, and positive and negative predictive values for the plasmid PCR in genital specimens were 100%, 98%, 95.7%, and 100%, respectively. Corresponding values for urine PCR were 81.8%, 100%, 100%, and 92.5%, respectively. The prevalence of confirmed C. trachomatis infection was 30.8% in this STD population. The results confirmed the need to use sensitive and specific molecular assays like PCR to prevent underdiagnosis of genital chlamydial infections and to facilitate better clinical management of this infection in India.