ABSTRACT Staphylococcus epidermidis is a major cause of catheter-related bloodstream infections (CRBSIs). Recent studies suggested the existence of well-adapted, highly resistant, hospital-associated S. epidermidis clones. The molecular epidemiology of S. epidermidis in Belgian hospitals and the Belgian community has not been explored yet. We compared a set of 33 S. epidermidis isolates causing CRBSI in hospitalized patients with a set of 33 commensal S. epidermidis isolates. The factors analyzed included resistance to antibiotics and genetic diversity as determined by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and SCC mec typing. Additionally, the presence of virulence-associated mobile genetic elements, the ica operon and the arginine catabolic mobile element (ACME), was assessed and compared against clinical data. CRBSI S. epidermidis isolates were significantly resistant to more antibiotics than commensal S. epidermidis isolates. The two populations studied were very diverse and genetically distinct as only 23% of the 37 PFGE types observed were harbored by both CRBSI and commensal isolates. ACME was found in 76% of S. epidermidis strains, regardless of their origin, while the ica operon was significantly more prevalent in CRBSI isolates than in commensal isolates ( P < 0.05). Nine patients presented a clinically severe CRBSI, eight cases of which were due to an ica -positive multiresistant isolate belonging to sequence type 2 (ST2) or ST54. S. epidermidis isolates causing CRBSI were more resistant and more often ica positive than commensal S. epidermidis isolates, which were genetically heterogeneous and susceptible to the majority of antibiotics tested. Clinically severe CRBSIs were due to isolates belonging to two closely related MLST types, ST2 and ST54.