Chlamydia, with its Chlamydia trachomatis etiology, is the most common bacterial sexually transmitted infection in the United States and is often transmitted via asymptomatic individuals. This review summarizes traditional and molecular-based diagnostic modalities specific to C. trachomatis. Several commercially available, FDA-approved molecular methods to diagnose urogenital C. trachomatis infection include nucleic acid hybridization, signal amplification, polymerase chain reaction, strand displacement amplification, and transcription-mediated amplification. Molecular-based methods are rapid and reliable genital specimen screening measures, especially when applied to areas of high disease prevalence. However, clinical and analytical sensitivity for some commercial systems decreases dramatically when testing urine samples. In vitro experiments and clinical data suggest that transcription-mediated amplification has greater analytical sensitivity than the other molecular-based methods currently available. This difference may be further exhibited in testing of extragenital specimens from at-risk patient demographics. The development of future molecular testing could address conundrums associated with confirmatory testing, medicolegal testing, and test of cure.