The presence of Salmonella enterica throughout the production and processing continuum is a concern in broiler industry. While federal regulations have lowered the acceptable level of Salmonella contamination on broiler carcasses, the mechanisms that contribute to pathogen attachment are not fully understood. Salmonella Kentucky has become the predominant Salmonella serovar isolated from broilers carcasses at the end of the immersion chill tank. In Europe and Africa this serovar has been shown to acquire antibiotic resistance genes that may lead this non-typhoidal serotype to become a potential public health concern. To investigate the genes that are involved in colonization of the bacteria to broiler skin, a mutant library of the bioluminescent strain of S. Kentucky was constructed. According to the chicken attachment assay, it was concluded that attachment is a multifactorial process with the following elements contributing: i), flagella, ii), LPS structure, iii), amino acid metabolism, iv), TCA cycle pathway; v), conjugative transfer system, vi), multidrug resistant protein, vii), signaling and transportation system, viii), metabolism, ix), different enzymes, x), phage tail fiber protein H, xi), fimbrial export usher proteins, xii), membrane proteins xiii), and several unnamed proteins. The role of flagella between all of these contributing elements appeared to be the most significant. The flagella motor gene, filament sub-units and hook associated protein were deleted by using the ë red recombination method. The mutants’ ability to colonize broiler skin was compared to their parental strain, and the motility and flagellin main sub-unit (FliC) were recognized as the key factors contributing to bacterial attachment. Using Caco-2 cell lines as a cell model to assess adhesion and invasion capacity of flagella mutants, similar results were observed. Based on the result of the experiments conducted in this study, it appears that the active flagella FiC sub-unit plays an important role in colonization of epithelial cells outside and inside of the broilers.