Molecular diagnostics of infectious diseases, in particular, nucleic-acid-based methods, are the fastest growing field in clinical laboratory diagnostics. These applications are stepwise replacing or complementing culture-based, biochemical, and immunological assays in microbiology laboratories. The first-generation nucleic acid assays were monoparametric such as conventional tests, determining only a single parameter. Improvements and new approaches in technology now open the possibility for the development of multiparameter assays using microarrays, multiplex nucleic acid amplification techniques, or mass spectrometry, while the introduction of closed-tube systems has resulted in rapid microbial diagnostics with a subsequently reduced contamination risk. Whereas the first assays were focused on the detection and identification of microbial pathogens, these new technologies paved the way for the parallel determination of multiple antibiotic resistance determinants or to perform microbial epidemiology and surveillance on a genetic level.