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Mastitis is the most common and detrimental infection of the mammary gland in dairy cows and has a major economic impact on the production of milk and dairy products. Bacterial mastitis is caused by several pathogens, and the most frequently isolated bacterial species are coagulase-negative staphylocci (CNS). Although CNS are considered minor mastitis pathogens, the importance of CNS has increased over the years. However, the mechanism and factors involved in CNS intramammary infection are poorly studied and defined. Biofilms have been proposed as an important component in the persistence of CNS intramammary infection. Biofilms are defined as a cluster of bacteria enclosed in a self-produced matrix. The objectives of this study were to investigate the ability of CNS to form biofilms. A total of 255 mastitis-associated CNS isolates were investigated using a standard microtiter plate biofilm assay. The biofilms of some isolates were also observed by using confocal microscopy. The presence of biofilm-associated genes icaA, bap, aap, embP, fbe, and atlE was determined by PCR in the 255 isolates. The 5 dominant species assayed were Staphylococcus chromogenes (n=111), Staphylococcus simulans (n=53), Staphylococcus xylosus (n=25), Staphylococcus haemolyticus (n=15), and Staphylococcus epidermidis (n=13), and these represented 85% of the isolates. The data gathered were analyzed to identify significant links with the data deposited in the Canadian Bovine Mastitis Research Network database. Overall, Staph. xylosus is the species with the strongest ability to form biofilm, and Staph. epidermidis is the species with the lowest ability to form biofilm. Regardless of the species, the presence of icaA, bap, or the combination of multiple genes was associated with a greater ability to form biofilm. A strong relationship between the strength of a biofilm and days in milk was also noted, and CNS isolated later in the lactation cycle appeared to have a greater ability to form biofilm than those isolated earlier in the lactation cycle. In conclusion, Staph. xylosus is the species with the strongest biofilm formation ability. Furthermore, days in milk and gene combinations are predicted to be the variables with the strongest effect on biofilm formation by CNS.