ObjectiveTo investigate the homology of methicillinresistant Stphylococcus aureus(MRSA) from the neonatal intensive care unit(NICU) of a children’s hospital, and evaluate routes and preventive strategies of MRSA healthcareassociated infection(HAI). MethodsMRSA strains from neonates and environment of NICU between October and December 2014 were collected, and strains were identified by VITEK2 microbial analysis system and cefoxitin KirbyBauer method, homology of MRSA was analyzed by pulsedfield gel electrophoresis (PFGE). ResultsA total of 6 MRSA strains were isolated from NICU between October and December 2014, 3 of which (bed58, 70, and 100) were detected MRSA from specimens, MRSA were isolated from neonatal incubator and nurse (nasal swabs and hands) who cared for neonate at bed 58. 5 of 6 MRSA strains were homology, antimicrobial susceptibility testing result showed that No. 1-5 strains were resistant to clindamycin and amoxicillin/clavulanic acid, No. 6 strain was slightly different from No. 1-5 strains, No. 6 strain was susceptible to both clindamycin and amoxicillin/clavulanic acid. PFGE results showed that No. 1-5 strains were of the same type, No. 6 strain was a different type. ConclusionThe main route of this MRSA transmission is contact transmission, especially through the hands of health care workers, identification and analysis of epidemic strains by PFGE technique is an effective measures to prevent HAI outbreak and perform epidemiological study