Nosocomial infections among infants in neonatal intensive care units are an increasing cause of morbidity and mortality. Efforts to rapidly detect, contain and ultimately prevent cross-transmission of these pathogens require constant vigilance by members of the epidemiology team. A vital component of an epidemiological evaluation is the ability to assess the interrelatedness of clinical isolates. This review focuses on the basic principles, strengths and weaknesses of some of the most commonly used molecular-based procedures, which determine clonal identity of outbreak strains in neonatal settings. Published evidence for the cogent selection of the method best suited for studying a particular organism and technological advances that await the new science of Molecular Epidemiology are also presented. The appropriate use of molecular typing technologies should enable reliable tracking of epidemic clones and thereby enhance the effectiveness of infection control strategies.