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Fast and adequate detection of extended-spectrum β-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M.Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various β-lactamases were included (n = 106 ESBL positive).The sensitivity of the microarray was 101/106 (95%; 95% CI 89%–98%), and the specificity 100% (95% CI 97%–100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates.This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.