Salmonella Enteritidis has been a leading cause of foodborne illnesses worldwide, and food contamination continue to occur despite implemented foodborne pathogen reduction interventions. A key factor that might contribute to Salmonella persistence is the formation of biofilms, which enhance Salmonella’s resilience. This study aims to evaluate the biofilm formation abilities of Salmonella Enteritidis (SE) and Salmonella Kentucky (SK) at 20-22°C, linking phenotypic date with genomic insights through comparative analysis of marker genes associated with biofilm formation and strain resilience. Fifteen SE and 24 SK strains isolated from poultry processing facilities were compared to assess their biofilm-forming abilities. Biofilm were formed for 5 d at 20-22°C and assessed using the crystal violet method (CV). Genomes were sequenced using Illumina, assembled and annotated using the BV-BRC pipeline. A comparative systems analysis was conducted to screen for genes associated with biofilm formation. A protein-coded gene comparison was conducted to compare the only SE intermediate bovine strain to a strong SE poultry strain. Single Nucleotide Polymorphism (SNP) variation analysis was used to screen for genetic mutations. There were 11 strong and 4 intermediate SE strains, and 4 strong, 5 weak, and 15 non-biofilm SK strains. For curli and cellulose production, 11/15 SE and 4/24 SK strains were positive for both. A pan-genome of 39 Salmonella strains generated 5,396 genes and 122 of them were associated with biofilm formation, curli, cellulose, fimbria, flagellum, pilus, and type IV secretion system (T4SS) that might impact biofilm formation, and 107 of them were common to all strains. Strains resulted in almost the same genomic profiles except for curli genes csgDEF and T4SS genes virB1, virB3, virB4, virB6, virB10, virB11, and virD4. Twenty-four genes with mutations could possibly influence the biofilm formation, including bcsFGQ, flgAB, motAB, and tolABR. The nonsynonymous mutations of the bcs and tol genes might play important roles in biofilm forming capacity, explaining the weaker biofilm within some Salmonella strains. Protein-coded gene comparison revealed the absence of some T4SS proteins, including VirB1, VirB3, VirB4, VirB5, VirB6, VirB9, VirB10, VirB11, TraR (VirD2 homolog), and VirD4 in the intermediate strain while present in the strong biofilm strain.