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Biofilm formation is crucial for the environmental survival and transmission of Vibrio parahaemolyticus , an important food-borne pathogen in seafood. The biofilm developmental process of pathogenic (n = 22) and non-pathogenic (n = 17) V. parahaemolyticus strains on polystyrene microtiter plates under 15 °C, 25 °C and 37 °C was investigated using crystal violet staining, and validated by confocal laser scanning microscopy. The results indicated that biofilm developmental process at 15 °C and 25 °C were divergent, biofilm formation increased continuously at 15 °C, while at 25 °C biofilm formation increased gradually and peaked at 12 h. Also the biofilm formation was dramatically elevated at 25 °C in comparison with that at 15 °C and 37 °C. Additionally, pathogenic strains, on average, formed more biofilm than non-pathogenic strains at all temperatures measured. Moreover, extensive strain variability was observed during biofilm formation and indexed using the coefficient of variation (CV). This index increased with increasing temperature and this index, at all temperatures, peaked after 12 h. The results of this study provide insight into the developmental process of biofilm, which allow us to further optimize strategies to control V. parahaemolyticus biofilm in food industry. [ABSTRACT FROM AUTHOR]