The use of nucleic acid (NA) amplification techniques has transformed the diagnosis of viral infections of the central nervous system (CNS). Because of their enhanced sensitivity, these methods enable detection of even low amounts of viral genomes in cerebrospinal fluid. Following more than 10 years of experience, the polymerase chain reaction or other NA-based amplification techniques are nowadays performed in most diagnostic laboratories and have become the test of choice for the diagnosis of several viral CNS infections, such as herpes encephalitis, enterovirus meningitis and other viral infections occurring in human immunodeficiency virus-infected persons. Furthermore, they have been useful to establish a viral etiology in neurological syndromes of dubious origin and to recognise unusual or poorly characterised CNS diseases. Quantitative methods have provided a valuable additional tool for clinical management of these diseases, whereas post-amplification techniques have enabled precise genome characterisation. Current efforts are aiming at further improvement of the diagnostic efficiency of molecular techniques, their speed and standardisation, and to reduce the costs. The most relevant NA amplification strategies and clinical applications of to date will be the object of this review.