Tuberculosis is the predominant infectious cause of mortality today, killing 3 million people annually. The cornerstones of diagnosis rest on microscopy of specimens using auramine and Ziehl-Neelsen stains followed by culture on Lowenstein-Jensen or alternative media. The long generation time of Mycobacterium tuberculosis means 2-8 weeks usually elapse before a result is available to the clinician. This has stimulated research into the use of molecular diagnostic techniques. This article reviews the use and limitations of DNA hybridization, restriction fragment length polymorphism, pulsed-field gel electrophoresis and the polymerase chain reaction in the diagnosis and epidemiology of tuberculosis. The applicability of molecular biology to determine drug resistance is also addressed.