Listeria monocytogenes can cause serious illness in humans, usually following the ingestion of contaminated food. Epidemiologic investigation requires identification of specific isolates, usually done by a combination of serotyping and subtyping using pulsed-field gel electrophoresis (PFGE). DNA microarrays provide a new format to resolve genetic differences among isolates and, unlike PFGE, to identify specific genes associated with the infecting pathogen. A 585 probe, mixed genome microarray was constructed and 24 strains of L. monocytogenes were hybridized to the array. Microarray analysis allowed discrimination among L. monocytogenes isolates within a serotype and obtained from similar geographic and epidemiologic sources. Importantly, the microarray results preserved previously described phylogenetic relationships between major serogroups and, in a limited comparison, agreed with PFGE subtypes. The association of individual probes with isolates allowed identification of specific genes. Sequencing of 10 polymorphic probes identified nine matches with previously described bacterial genes including several suspected virulence factors. These results demonstrate that mixed genomic microarrays are useful for differentiating among closely related L. monocytogenes isolates and identifying genetic markers that can be used in epidemiologic and possibly pathogenesis studies.