Purpose: To determine utility of polymerase chain reaction (PCR)-based urine screening for Chlamydia trachomatis in the care of adolescent females in an urban clinic. Methods: Females ≤15 years of age attending an adolescent clinic were approached consecutively. Each enrollee was interviewed to determine the primary reason(s) for the clinic visit and was queried about genitourinary symptoms. Nonsterile voided urine specimens were tested for C. trachomatis using PCR-based analysis. Endocervical C. trachomatis cultures were obtained from the subjects who had a pelvic examination. Main outcome measures were chlamydia infection rates in clinic attendees whether a pelvic examination was performed or not. Results: A total of 315 (99.4%) of 317 patients approached agreed to participate. Overall, 47 (14.9%) patients had positive urine PCR tests. The chlamydia infection rate detected by urine PCR was 22.1% (19 of 86) among those who had pelvic examinations performed and 12.2% (28 of 229) among those who did not (p = .03; odds ratio 2.04; 95% confidence interval 1.02, 4.06). Sixty percent (28 of 47) of chlamydia infections identified during the study period were identified by the urine screening test. Conclusion: Urine screening was accepted by vast majority of female adolescents attending the clinic irrespective of reason for the clinic visit, and was highly effective in identifying unsuspected C. trachomatis infections, particularly among girls attending the clinic for reasons unrelated to reproductive health care and as an interim screening tool for adolescent family-planning clients.