Coagulase-negative staphylococci (CoNS) are the main cause of catheter-related infections, especially among immunosuppressed and neutropenic patients, as well as a source of bacterial contamination in blood cultures. Using biochemical identification and pulsed-field gel electrophoresis (PFGE), we sought to identify possible clonal isolates of bacteremia in patients with central lines in an oncology ward (OW), with comparison to isolates that were recovered by venipuncture from an adult emergency room (ER). A total of 243 CoNS isolates were identified to species level from the OW (126) and ER (117), with Staphylococcus epidermidis isolates being the most common (OW, 79.4%; ER, 45.3%). PFGE demonstrated a predominant clone of S. epidermidis (major subtype A) which was 35.5 times more likely (odds ratio [OR] = 35.5; 95% confidence interval [CI] = 4.7 to 267.0; P < 0.00001) to be present in the OW versus the ER. These (CoNS or major subtype A) isolates were more frequently resistant to gentamicin (OR = 2.83; 95% CI = 1.23 to 6.53; P = 0.016) and less frequently resistant to trimethoprim-sulfamethoxazole (OR = 0.38; 95% CI = 0.18 to 0.80; P = 0.013). Subset analysis of S. epidermidis isolates 2 years after the study period showed the persistence of the clone of major subtype A within the OW. This study demonstrates the presence of a predominant clone among central line isolates from an OW that is not present in CoNS venipuncture isolates from an ER.