Listeria monocytogenes is of great concern as a foodborne pathogen. Many ready-to-eat foods are widely contaminated with this organism and have caused listeriosis outbreaks and sporadic cases in many countries. In Japan, there is a high incidence of L. monocytogenes contamination, specifically in raw ready-to-eat seafood. Identical L. monocytogenes subtypes have been isolated repeatedly from samples of food manufactured at a given store or processing plant, and researchers suspected that certain L. monocytogenes isolates have formed biofilms at these sites. A microtiter plate biofilm formation assay was conducted, and all raw ready-to-eat seafood isolates tested were able to form biofilms to various degrees. Biofilm formation by L. monocytogenes isolates of lineage I was significantly greater (P = 0.000) than that by isolates of lineage II. However, isolates of clonal lineages formed different levels of biofilms, indicating that the ability to form a biofilm is affected positively or negatively by environmental factors.