Objectives To assess the frequency and diversity of extended-spectrum β-lactamases (ESBLs) in Enterobacteriaceae isolates in Belgium. Methods During 2006 and 2008, non-duplicate clinical isolates of Enterobacteriaceae resistant to ceftazidime and/or cefotaxime were collected in 100 Belgian hospitals. ESBL production was confirmed by phenotypic and genotypic tests. MICs of 13 antimicrobial agents were determined by Etest. ESBL-encoding genes were identified by PCR sequencing and the bla CTX-M environment was characterized by PCR mapping. Selected isolates were genotyped by PFGE, multilocus sequence typing analysis and phylogenetic grouping by PCR. Results Overall, 733 isolates were confirmed as ESBL producers. Carbapenems and temocillin were active against ≥95% of all tested isolates. Co-resistance to co-trimoxazole and to ciprofloxacin was found in almost 70% and 80% of the strains, respectively. Overall, Escherichia coli (49%), Enterobacter aerogenes (32%) and Klebsiella pneumoniae (9%) represented the most prevalent species. Isolates harboured predominantly TEM-24 (30.7%), CTX-M-15 (24.2%) and TEM-52 (12.1%). Compared with 2006, the proportion of CTX-M-type enzymes increased significantly in 2008 (54% versus 23%; P < 10−6), mostly linked to a rising proportion of CTX-M-15-producing E. coli . TEM-24 decreased (19% in 2008 versus 43% in 2006; P < 10−6) during the same period, while the prevalence of TEM-52 remained unchanged (10% in 2008 versus 14% in 2006; not significant). Over 80% of the CTX-M-15-producing E. coli isolates clustered into a single PFGE type and phylogroup B2, corresponding to the sequence type (ST) 131 clone. Intra- and inter-species gene dissemination (CTX-M-15, CTX-M-2 and CTX-M-9) and wide epidemic spread of the CTX-M-15-producing E. coli ST131 clone in several Belgian hospitals were observed. Conclusions The rapid emergence of multiresistant CTX-M-15-producing E. coli isolates is of major concern and highlights the need for further surveillance in Belgium.