Technical Abstract: Contamination of food by Listeria monocytogenes is thought to occur most frequently in food processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells produce multi-cellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and virulence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation was also detected between persistent and nonpersistent strains isolated from bulk milk samples with persistent strains showing increased biofilm formation. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high biofilm producing strains. Scanning electron microscopy showed that a high biofilm forming strain produced a dense, three-dimensional structure whereas, a low biofilm forming strain produced a thin, patchy biofilm. These data are consistent with persistent strains forming biofilms, but do not support a consistent relationship between enhanced biofilm formation and virulence.