Coagulase-negative staphylococcus (CNS) is the most frequent cause of nosocomial bacteremia and prosthetic valve endocarditis. CNS bacteremia can be polyclonal. No data exist on the clonality of CNS causing endocarditis. We present a case of CNS aortic homograft endocarditis in which at least five different genotypes of CNS were identified in initial blood-culture isolates by genomic macrorestriction enzyme analysis and pulsed field gel electrophoresis. Since the polyclonality was accompanied by differences in antibiotic susceptibility, this observation may have important consequences for the treatment of CNS endocarditis. Because of the parallels in the pathogenesis of CNS prosthetic valve endocarditis and CNS infections of a variety of other prosthetic devices, it might also have consequences for CNS prosthetic device infections in general. We suggest that antibiotic susceptibility testing of just one blood-culture isolate may be insufficient.