AbstractObjective:To investigate the molecular epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) in infants in a neonatal intensive care unit (NICU) using whole-genome sequencing.Design:Investigation of MSSA epidemiology in a NICU.Setting:Single-center, level IV NICU.Methods:Universal S. aureus screening was done using a single swab obtained from the anterior nares, axilla, and groin area of infants in the NICU on a weekly basis. Core genome multilocus sequence type (cgMLST) analysis was performed on MSSA isolates detected over 1 year (2018–2019).Results:In total, 68 MSSA-colonized infants were identified, and cgMLSTs of 67 MSSA isolates were analyzed. Overall, we identified 11 cgMLST isolate groups comprising 39 isolates (58%), with group sizes ranging from 2 to 10 isolates, and 28 isolates (42%) were unrelated to each other or any of the isolate groups. Cases of infants colonized by MSSA were scattered throughout the 1-year study period, and isolates belonging to the same cgMLST group were typically detected contemporaneously, over a few weeks or a few months. Overall, 13 infants (19.7%) developed MSSA infections: bacteremia (n = 3), wound infection (n = 5), conjunctivitis (n = 4), and cellulitis (n = 1). We detected no association between these clinically manifest infections and specific cgMLST groups.Conclusions:Although MSSA isolates in infants in a NICU showed high diversity, most were related to other isolates, albeit within small groups. cgMLST facilitates an understanding of the complex transmission dynamics of MSSA in NICUs, and these data can be used to inform better control strategies.