Molecular diagnostic tests are more sensitive and, in many cases, more specific than conventional laboratory methods for the detection of sexually transmitted infections. Here, we review recently developed molecular methods for the diagnosis and subtyping of the most common sexually transmitted infections: infections caused by Chlamydia trachomatis, Neisseria gonorrhoeae, human papillomavirus, Trichomonas vaginalis, and the agents of genital ulcer disease (Haemophilus ducreyi, herpes simplex virus, Treponema pallidum, and Calymmatobacterium granulomatis). We also provide an overview of the laboratory diagnostic tests and clinical specimens to use when infection with these agents is suspected.