Abstract: The global prevalence and spread of multidrug-resistant organisms (MDROs) represent an emerging public health threat. Daycare center (DCC) attendance is a risk factor for MDRO carriage in children and their environment. This study aimed to map the epidemiology of carriage and potential transmission of these organisms within 18 Flemish DDCs (Belgium). An MDRO prevalence survey was organized between November 2018 and February 2019 among children attending the centres. Selective chromogenic culture media were used for detection of extended-spectrum beta-lactamase producing Enterobacterales (ESBL-E), carbapenemase producing Enterobacterales (CPE) and vancomycin resistant Enterococci (VRE) in faecal swabs obtained from diapers or jars (n=448). All isolated MDROs were subjected to resistance gene sequencing. A total of 71 out of 448 samples (15.8%) yielded isolates of ESBL-E with a predominance of Escherichia coli (92.2% of ESBL-E) and ESBL resistance gene blaCTX-M-15 (50.7%) of ESBL coding genes in E. coli). ESBL-E prevalence varied between DCCs, ranging from 0 to 50%. Transmission, based on clonal relatedness of ESBL-E strains, was observed. CPE was identified in only one child carrying an E. coli with an OXA-244 gene. VRE was absent from all samples. The observed prevalence of ESBL-E in Flemish DCCs is high compared to previous studies and our findings re-emphasize the need for rigorous hygiene measures within such centres to control further spread of MDROs in the community.