Listeria monocytogenes is among the most important food-borne pathogens and is well adapted to persist in the environment. To gain insight into the genetic relatedness and potential virulence of L. monocytogenes strains causing central nervous system (CNS) infections, we used multilocus variable-number tandem-repeat analysis (MLVA) to subtype 183 L. monocytogenes isolates, most from ruminant rhombencephalitis and some from human patients, food, and the environment. Allelic-profile-based comparisons grouped L. monocytogenes strains mainly into three clonal complexes and linked single-locus variants (SLVs). Clonal complex A essentially consisted of isolates from human and ruminant brain samples. All but one rhombencephalitis isolate from cattle were located in clonal complex A. In contrast, food and environmental isolates mainly clustered into clonal complex C, and none was classified as clonal complex A. Isolates of the two main clonal complexes (A and C) obtained by MLVA were analyzed by PCR for the presence of 11 virulence-associated genes (prfA, actA, inlA, inlB, inlC, inlD, inlE, inlF, inlG, inlJ, and inlC2H). Virulence gene analysis revealed significant differences in the actA, inlF, inlG, and inlJ allelic profiles between clinical isolates (complex A) and nonclinical isolates (complex C). The association of particular alleles of actA, inlF, and newly described alleles of inlJ with isolates from CNS infections (particularly rhombencephalitis) suggests that these virulence genes participate in neurovirulence of L. monocytogenes. The overall absence of inlG in clinical complex A and its presence in complex C isolates suggests that the InlG protein is more relevant for the survival of L. monocytogenes in the environment.