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Staphylococcus saprophyticus is an important agent of urinary tract infection (UTI) in young women, but information about this pathogen in human microbiota and in common environment is lacking. The aim of this study was to characterize S. saprophyticus isolates from genitoanal microbiota of 621 pregnant women, 10 minas cheese packs, and five beaches in Rio de Janeiro city and compare PFGE profiles of these isolates with five UTI PFGE clusters described in this city. We investigated 65 S. saprophyticus isolates from microbiota, 13 from minas cheese, and 30 from beaches and 32 UTI isolates. Antimicrobial resistance was determined by disk diffusion, MIC by agar dilution, and PCR. Erythromycin-resistance genes erm(C), msr(A), msr(B), mph(C), and lin(A) were found in 93% of isolates. Trimethoprim-sulfamethoxazole resistance correlated with dfrG or dfrA genes. Three cefoxitin-resistant isolates carried the mecA gene. All isolates obtained from cheese were susceptible to all antimicrobial agents. Six of 10 pregnant women with >1 isolate had monoclonal colonization. Isolates from pregnant women shared 100% similarity with UTI PFGE cluster types A and E obtained almost 10 years previously, suggesting temporal persistence of S. saprophyticus. Antimicrobial resistance of beach isolates reflected the profiles of human isolates. Taken together, results indicate a shared source for human and environmental isolates.