Methicillin-resistant Staphylococcus aureus (MRSA) represent a serious threat for public health worldwide. Of particular concern is the emergence of community-acquired MRSA, which is often difficult to distinguish from nosocomial MRSA due to a lack of suitable typing methods for early detection. For example, the USA300 pulsed-field gel electrophoresis (PFGE) pattern includes both the 'classical' community-acquired USA300 clone with spa type t008 and an epidemiologically unrelated nosocomial clone with spa type t024. Likewise, spa typing cannot distinguish the classic USA300 from nosocomial MRSA with the spa type t008. Since the fast and high-resolution distinction of these S. aureus types is important for infection prevention and surveillance, we investigated whether multiple-locus variable number tandem repeat fingerprinting (MLVF) can be applied to overcome these limitations. Indeed, MLVF correctly grouped 91 MRSA isolates belonging to the classic USA300 lineage, nosocomial MRSA isolates with the USA300 PFGE profile and spa type t024, and nosocomial MRSA isolates with spa type t008 into 3 distinct clusters. Importantly, several sub-clusters were also identified, reflecting epidemiological relationships between the respective isolates. We conclude that MLVF has the discriminatory power needed to rapidly distinguish very similar community-acquired and nosocomial MRSA isolates and that MLVF-based sub-clustering of isolates is highly useful for epidemiological investigations, outbreak prevention, and control. (C) 2012 Elsevier GmbH. All rights reserved.