One of the criteria used to determine the clinical importance of coagulase-negative staphylococci (CoNS) is isolation of the bacteria from sequential blood cultures. Pulsed-field gel electrophoresis was used to characterize sequential blood isolates of CoNS collected within a 7-day period from neonates and children with bacteremia. Of 18 episodes among neonates, 6 (33%) involved unrelated strains of CoNS. All unrelated strains were from neonates who received antimicrobial therapy after the first culture and who had a second culture ≤36 h later. Among older children, 5 (19%) of the 27 episodes of presumed central venous (CV) catheter-related sepsis involved unrelated isolates. All of the unrelated isolates were from patients who had blood samples obtained through CV catheters only. Thus, even repeated isolation of CoNS from blood cultures may represent contamination if samples are drawn through CV catheters only or if second samples are obtained >1 day after appropriate antimicrobial therapy.