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OBJECTIVES The aim of this study was to investigate the carriage rates of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) among healthcare workers (HCWs) at a tertiary-care hospital in southeastern China and to analyse the epidemiological relatedness of the S. aureus isolates. METHODS A total of 242 pharynx swabs were collected in March-April 2016 from 242 HCWs working in seven different wards and departments. Isolates were identified as S. aureus based on morphology, coagulase test and Vitek test. Antimicrobial susceptibility testing was performed by using the Kirby-Bauer disk diffusion method. The epidemiological relatedness of the S. aureus isolates was determined by pulsed-field gel electrophoresis (PFGE). RESULTS From the 242 HCWs, 70 (28.9%) S. aureus strains, including 10 (4.1%) MRSA strains, were identified during screening, with the highest MRSA rate detected in nurses (8/107; 7.5%). Carriage rates of S. aureus and MRSA among surgical HCWs were 30.0% (63/210) and 4.3% (9/210), respectively. Methicillin-susceptible S. aureus (MSSA) isolates were grouped by PFGE analysis into five similar groups (A-E), with most isolates belonging to groups D and E, accounting for 63.0% of isolates. Furthermore, two MRSA isolates from gastrointestinal surgery had identical PFGE patterns. CONCLUSIONS The prevalence of S. aureus colonisation among HCWs was high in this hospital, although the MRSA carriage rate in surgical wards was low. The identical PFGE pattern detected in two MRSA isolates from the same surgical department supports that effective control for possible cross-infection should be implemented.