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is a clinically significant, opportunistic pathogen adept at thriving in both host-associated and environmental settings. We sought to define the extent to which isolates specialize across niches using a comprehensive study of whole-genome sequencing with paired phenotypic characterization of 125 . isolates from diverse clinical and environmental sites. We evaluated virulence-associated traits, including motility, cytotoxicity, biofilm formation, pyocyanin production, and antimicrobial resistance to eight antibiotics. Our results show that genomic diversity does not correlate with isolation source or most virulence phenotypes. Instead, we find that, in agreement with prior studies, the two major clades (groups A and B) clearly segregate by cytotoxicity, with group B strains showing significantly higher cytotoxicity than group A. Sequence analysis revealed previously uncharacterized alleles of genes encoding type III secretion effector proteins. We observed high variability among strains and isolation sources in the four assayed virulence phenotypes. Antimicrobial resistance was exclusively observed in clinical isolates, whereas it was absent in environmental isolates, reflecting antibiotic exposure-driven selection. Bacterial genome-wide association studies (GWAS) revealed an association between cytotoxicity and presence, and we identified a novel allelic variant with decreased cytotoxicity, demonstrating that functional diversity of well-characterized virulence factors may influence pathogenic outcomes. Overall, our analysis supports the hypothesis that the ability of to thrive across diverse niches is driven not by niche-specific accessory genes but by its core genome. Thus, isolates are capable of broad niche colonization without initial genetic adaptations.IMPORTANCE is a clinically significant opportunistic pathogen adept at thriving in both host-associated and environmental niches. A major gap in our understanding of this difficult-to-treat pathogen is whether niche specialization occurs in the context of human disease. Addressing this question is critical for guiding effective infection control strategies. Previous large-scale studies have focused solely on genotypic or phenotypic analyses; when paired, they have been limited to a single phenotypic assay or to a small number of isolates from one source, or relied on PCR-based methods targeting a restricted set of genes. To comprehensively uncover niche specialization and pathogenic versatility, we performed whole-genome sequencing and phenotypic characterization of five virulence-associated traits, including antimicrobial susceptibility of 125 clinical and environmental isolates. Our systems-level findings challenge reductionist models of bacterial niche specialization, instead supporting an integrated view where conserved genomic systems enable opportunistic pathogenesis across diverse environments.