In principle, restriction fragment length polymorphism (RFLP) typing can be applied to strains of all mycobacterial species for which suitable probes have been identified. International consensus has been achieved regarding the methodology of IS6110 RFLP typing of Mycobacterium tuberculosis complex isolates (1 ) and IS1245 RFLP typing of Mycobacterium avium strains (2 ). This chapter describes the technical details of these standardized methods regarding the isolation of DNA, restriction enzymes, electrophoresis conditions, internal- and external-size markers, Southern blotting, and several probes used for hybridization. Furthermore, RFLP typing of isolates of some other mycobacterial species is described.