Some Listeria monocytogenes strains, termed persistent strains, originate from the same processing plant and have the ability to survive and grow over extended periods of time at contamination sources. In order to evaluate biofilm formation by such persistent strains, we isolated the pathogen from chicken samples collected from the same retail shop in repeated visits over 6 months. Strains that were of serotype 1/2b and were assigned to the same genotype by multi-virulence-locus sequence typing analysis were isolated on repeated occasions from December 1997 to June 1998 and thus were defined as persistent strains. In the present study, biofilm formation by the persistent strains was evaluated using microplates at 30 and 37°C. The biofilm-forming capability was measured after cells attaching to the microplate well were stained with crystal violet. Comparison of biofilm formation at 30°C among the persistent strains showed that a significantly higher amount of the stain was obtained from the persistent strains isolated from December to March than from those isolated from April to June. However, no significant difference in biofilm formation at 30°C was observed between persistent and nonpersistent groups of L. monocytogenes strains. In contrast, biofilm formation at 37°C was consistent among the persistent strains, and they produced significantly more biofilm at 37°C than did the nonpersistent strains. The persistent strains were also found to change their biofilm-forming ability in a temperature-dependent manner, which may suggest that the persistent strains alter their biofilm formation in response to changing environmental factors.