Background: Diagnosis of genital Chlamydia trachomatis infection in women traditionally requires a speculum examination to collect endocervical cells, followed by cell culture. This method is time consuming, requires stringent transport conditions, and is technically demanding. Goals: To compare tampons as a patient-administered collection method followed by detection with polymerase chain reaction (PCR) with the traditional endocervical swab culture followed by cell culture detection. Study Design: At the emergency department of a hospital for obstetrics and gynecology, 1,000 consecutive women with symptoms suggestive of infection with C. trachomatis were tested for C. trachomatis infection by PCR on both tampon (PCR-T) and swab (PCR-S) specimen and by culture of the swab specimen. Results: Seventeen PCR-T and 16 PCR-S specimens were positive; 16 endocervical specimens were positive by culture, and 14 of the endocervical samples were positive by the three methods. Sixty-one PCR-S samples were inadequate as shown by the lack of amplification of the β-globin gene segment, indicating poor collection of specimens by endocervical swab for chlamydial testing. Conclusions: Tampon specimens collected for PCR detection provided an easy and sensitive method of detection of C. trachomatis and overcame the obstacle of endocervical sampling and subsequent stringent transport requirements of culture.