Populations of the food-borne pathogen Listeria monocytogenes are genetically structured into a small number of major clonal groups, some of which have been implicated in multiple outbreaks. The goal of this study was to develop and evaluate an optimized multilocus variable number of tandem repeat (VNTR) analysis (MLVA) subtyping scheme for strain discrimination and clonal group identification. We evaluated 18 VNTR loci and combined the 11 best ones into two multiplexed PCR assays (MLVA-11). A collection of 255 isolates representing the diversity of clonal groups within phylogenetic lineages I and II, including representatives of epidemic clones, were analyzed by MLVA-11, multilocus sequence typing (MLST), and pulsed-field gel electropho-resis (PFGE). MLVA-11 had less discriminatory power than PFGE, except for some clones, and was unable to distinguish some epidemiologically unrelated isolates. Yet it distinguished all major MLST clones and therefore constitutes a rapid method to identify epidemiologically relevant clonal groups. Given its high reproducibility and high throughput, MLVA represents a very attractive first-line screening method to alleviate the PFGE workload in outbreak investigations and listeriosis surveillance. L isteriosis is a food-borne infection caused by the bacterium Listeria monocytogenes. Invasive forms of human listeriosis include septicemia, meningitis, and maternal-fetal infections (1). Listeriosis is associated with high hospitalization and fatality rates (almost 100% and 25 to 30%, respectively). Populations at risk include pregnant women, immunocompromised individuals, and the elderly. L. monocytogenes is widely present in the environment, including soil, water, vegetation, and silage, as well as in animals and animal-derived food, and can contaminate food in processing plants and retail establishments. L. monocytogenes is recognized as a public health issue and a serious challenge for the food industry, and this has led to the establishment of national surveillance systems in several countries. L. monocytogenes also stands out as a model system in the fields of microbiology, cell biology, and im-munology and for the study of host-pathogen interactions (2-5). L. monocytogenes strain characterization on the basis of sero-typing and molecular typing methods is used for surveillance, epidemiological tracking, and outbreak investigation purposes (6, 7). Genetic variants of L. monocytogenes have diversified into four major phylogenetic lineages, with lineages 1 and 2 each containing multiple clonal groups of public health importance (8-14). As these groups appear to differ in virulence and epidemic potential (6, 15), it will be interesting to better define their epidemiological, clinical, and microbiological specificities. For this purpose, tools for the easy identification of clonal groups are needed to recognize such groups and determine their presence in a large variety of sources. Several typing methods are currently available for L. monocytogenes strains. Conventional serotyping (16) and its molecular proxy PCR serogrouping (17) discriminate major categories of strains that correlate strongly (albeit not totally) with lin-eages and clones (11, 12, 14), but these methods do not have the necessary discriminatory power in the context of outbreak investigations. Pulsed-field gel electrophoresis (PFGE) is established as the gold standard for L. monocytogenes strain subtyping and is widely used for listeriosis surveillance and outbreak investigations (18). Yet, PFGE presents several practical disadvantages, as it is time-consuming and requires stringent standardization for inter-laboratory data comparison. Multilocus sequence typing (MLST) is a well-established reference method for global epidemiology and population biology (19, 20), as it renders interlaboratory genotype comparisons easy and unambiguous and as sequence data can be used to infer useful population genetic information such as amounts of genetic diversity, recombination rates, and strain phy-logeny. MLST also provides backward compatibility with genome sequencing (21). However, MLST is neither rapid nor cheap and has limited discriminatory power within L. monocytogenes (12, 22). Given the current limitations of available methods for L. monocytogenes strain typing, a potentially useful complementary approach is multilocus variable number of tandem repeats