Introduction The aim of this study was to determine the rate of asymptomatic carriage and spread of multidrug- resistant micro-organisms (MDRO) and to identify risk factors for extended spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in 12 long term care facilities (LTCFs) in Amsterdam, the Netherlands. Materials and methods From November 2014 to August 2015, feces and nasal swabs from residents from LTCFs in Amsterdam, the Netherlands were collected and analyzed for presence of multidrug-resistant Gram-negative bacteria (MDRGN), including ESBL-E, carbapenemase-producing Enterobacteriaceae (CPE), colistin-resistant Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Logistic regression analysis was performed to assess associations between variables and ESBLcarriage. Results In total, 385 residents from 12 LTCFs (range 15-48 residents per LTCF) were enrolled. The prevalence of carriage of MDRGN was 18.2% (range among LTCFs 0-47%) and the prevalence of ESBL-E alone was 14.5% (range among LTCFs: 0-34%). Of 63 MDRGN positive residents, 50 (79%) were ESBL-E positive of which 43 (86%) produced CTX-M. Among 44 residents with ESBL-E positive fecal samples of whom data on contact precautions were available at the time of sampling, only 9 (20%) were already known as ESBL-E carriers. The prevalence for carriage of MRSA was 0.8% (range per LTCF: 0-7%) and VRE 0%. One CPE colonized resident was found. All fecal samples tested negative for presence of plasmid mediated resistance for colistin (MCR-1). Typing of isolates by Amplified Fragment Length Polymorphism (AFLP) showed five MDRGN clusters, of which one was found in multiple LTCFs and four were found in single LTCFs, suggesting transmission within and between LTCFs. In multivariate analysis only the presence of MDRO in the preceding year remained a risk factor for ESBL-E carriage. Conclusions The ESBL-carriage rate of residents in LTCFs is nearly two times higher than in the general population but varies considerably among LTCFs in Amsterdam, whereas carriage of MRSA and VRE is low. The majority (80%) of ESBL-E positive residents had not been detected by routine culture of clinical specimens at time of sampling. Current infection control practices in LTCFs in Amsterdam do not prevent transmission. Both improvement of basic hygiene, and funding for laboratory screening, should allow LTCFs in Amsterdam to develop standards of care to prevent transmission of ESBL-E.