To gain a better understanding of epidemiology of resistance in Staphylococcus aureus, we describe the molecular epidemiology of methicillin-resistant Staphylococcus aureus bloodstream isolates in urban Detroit. Bloodstream isolates from July 2005 to February 2007 were characterized. Two hundred ten bloodstream isolates from 201 patients were evaluated. Patient characteristics were as follows: median age, 54 years; 56% male; and 71% African-American. Seventy-six percent of infections were health care associated, with 55% being community-onset infections and 21% hospital acquired, and 24% were community associated. The most common sources were skin/wound (25%), central venous catheters (24%), unknown source (20%), and endocarditis (9%). Ninety percent and 5% of isolates had a MIC of vancomycin of <or=1.0 mg/liter, using automated dilution testing and E-test, respectively. Six percent of isolates showed heteroresistance to vancomycin, all occurring with isolates having a vancomycin E-test MIC of >or=1.5 mg/liter. Results of pulsed-field gel electrophoresis showed 17 strain types. The predominant strains were USA100 (104 isolates) and USA300 (74 isolates). Forty-nine percent of the isolates had staphylococcal cassette chromosome mec II, and 56% had agr II. All USA300 isolates were positive for the Panton-Valentine leukocidin toxin genes and agr I. Forty-seven percent of USA300 bloodstream infections were health care associated (35% community onset and 12% hospital onset). USA300 strains were more common in injection drug users with skin/wound as the predominant source of infection. Thirty percent of the USA100 strains were closely related to vancomycin-resistant Staphylococcus aureus isolates. The results of this study show that vancomycin MICs using automated dilution testing with Vitek-2 and E-test were highly discordant. Most methicillin-resistant S. aureus strains causing bacteremia are health care associated, commonly have MICs of vancomycin that are high within the susceptible range are not detected by routine automated dilution testing, and have significant diversity of molecular characteristics. USA100 strains that are closely related to vancomycin-resistant S. aureus (VRSA) isolates and USA300 strains are common as causes of both hospital and community-onset infection. Infection control measures should focus not only on prevention of the spread of community strains in the hospital but also prevention of the spread of hospital strains associated with VRSA into the community.