Fifty-seven coagulase-negative Staphylococcus isolates from 22 inpatients who had > or =2 blood cultures that were positive for Staphylococcus within 24 hours were analyzed to determine the frequency of polyclonal bacteremia. Patients were considered to have bacteremia (14 patients) or contamination of sample (8 patients) on the basis of clinical criteria. Nine colonies were randomly selected from each blood culture and genotyped by means of SmaI digestion/pulsed-field gel electrophoresis. Relatedness was determined by calculation of the Dice coefficient of banding-pattern similarity (S(AB)). Analysis of bacteremic isolates demonstrated the presence of a single species in 35 of 41 blood cultures, 1 related variant in 5 blood cultures (87%-92% S(AB)), and an unrelated strain in 1 blood culture (79% S(AB)). Analysis of contaminated samples demonstrated the presence of a single strain in 10 of 16 blood cultures and 1-3 variants (28%-97% S(AB)) in the remainder. Genotype diversity was significantly more common in the contaminated samples (P=.036). Almost all coagulase-negative Staphylococcus bacteremias were monoclonal.