Enterococcus faecalisis the leading Gram-positive bacterial species implicated in urinary tract infection (UTI). An opportunistic pathogen,E. faecalisis a commensal of the human gastrointestinal tract (GIT) and its presence in the GIT is a predisposing factor for UTI. The mechanisms by whichE. faecaliscolonizes and survives in the urinary tract (UT) are poorly understood, especially in uncomplicated or recurrent UTI. The UT is distinct from the GIT and is characterized by a sparse nutrient landscape and unique environmental stressors. In this study, we isolated and sequenced a collection of 37 clinicalE. faecalisstrains from the urine of primarily postmenopausal women. We generated 33 closed genome assemblies and four highly contiguous draft assemblies and conducted a comparative genomics to identify genetic features enriched in urinaryE. faecaliswith respect toE. faecalisisolated from the human GIT and blood. Phylogenetic analysis revealed high diversity among urinary strains and a closer relatedness between urine and gut isolates than blood isolates. Plasmid replicon (rep) typing further underscored possible UT-GIT interconnection identifying nine shared rep types between urine and gutE. faecalis. Both genotypic and phenotypic analysis of antimicrobial resistance among urinaryE. faecalisrevealed infrequent resistance to front-line UTI antibiotics nitrofurantoin and fluoroquinolones and no vancomycin resistance. Finally, we identified 19 candidate genes enriched among urinary strains that may play a role in adaptation to the UT. These genes are involved in the core processes of sugar transport, cobalamin import, glucose metabolism, and post-transcriptional regulation of gene expression.IMPORTANCEUrinary tract infection (UTI) is a global health issue that imposes substantial burden on healthcare systems. Women are disproportionately affected by UTI with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. HowEnterococcus faecalis, a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinaryE. faecalisisolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may mediate urinaryE. faecalisadaptation to the female urinary tract.