Traditionally the detection of microbial pathogens in clinical, environmental or food samples has commonly needed the prelevation of cells by culture before the application of the detectionstrategy. This is done to increase cellnumber thereby overcoming problems associated with the sensitivity of classical detection strategies. However, culture-based methods have the disadvantages of taking longer, usually are more complex and require skilled personnel as well as not being able to detect viable but non cultivable microbial species. A number of molecular methods have been developed in the last 10 to 15 years to overcome these issues and to facilitate the rapid, accurate, sensitive and cost effective identification and enumeration of microorganisms which are designed to replace and/or support classical approaches to microbial detection. Amongst these new methods, ones based on the polymerase chain reaction and nucleic acid hybridization have been shown to be particularly suitable for this purpose. This review generally summarizes some of the current and emerging nucleic acid basedmolecular approaches for the detection, discrimination and quantification of microbes in environmental, food and clinical samples andincludes reference to the recently developing areas of microfluidics and nanotechnology “Lab-on-a-chip”.