The widespread presence of Listeria monocytogenes in diverse environments, including those that are natural (i.e., non-agricultural), agricultural, and foodassociated, suggests that these environments may serve as sources or reservoirs of L. monocytogenes that can be transmitted to various hosts, including humans. The vast majority of human listeriosis infections are recognized to occur through consumption of contaminated foods. Phylogenetic analysis of L. monocytogenes strains has identified three distinct lineages. While lineages I and II are both common among human clinical and food isolates, lineage I strains are overrepresented among clinical isolates, and lineage II strains are overrepresented among food and environmental isolates. Lineage III, which includes subgroups IIIA and IIIB, is rare and predominantly associated with animal disease. sigma-B, encoded by sigB, is a sigma factor previously demonstrated to critically contribute to stress response and virulence in lineage II strains. We used transcriptomic and phenotypic analyses to characterize the role of sigma-B in L. monocytogenes strains representing lineages I, II, IIIA, and IIIB. Whole-genome expression microarrays, phenotypic assays, and the guinea pig gastrointestinal model for listeriosis were used to characterize the role of sigma-B stationary phase wildtype and isogenic delta-sigB mutants representing L. monocytogenes diversity. Our results indicate that the role of sigma-B may differ among L. monocytogenes strains. The stationary phase transcriptome and sigma-B regulon of L. monocytogenes 10403S was also defined using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. We found that 83% of all genes were transcribed in stationary phase and a total of 96 genes had significantly higher transcript levels in 10403S than in delta-sigB, indicating sigma-Bdependent transcription of these genes. RNA-Seq analyses suggest that a total of 65 noncoding RNA molecules (ncRNAs) were transcribed in stationary phase. The RNA-Seq data also enabled annotation of putative operons and visualization of transcription start and stop sites. The results from these studies suggest that sigma-B contributes to a complex network of transcriptional regulators which allows L. monocytogenes to survive stress and subsequently cause disease and RNA-Seq allows quantitative characterization of prokaryotic transcriptomes and is a new strategy for exploring transcriptional regulatory networks in bacteria.