OBJECTIVES: We had for objective to measure the incidence and the clonal diversity of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBL) in order to assess the role of patient stay in amplification of the phenomenon, in our teaching hospital. MATERIAL AND METHODS: We measured the quarterly incidence rates of E. coli and K. pneumoniae producing or not producing ESBL in clinical samples between 1999 and 2010. The incidence of ESBL-producing isolates was season-adjusted. We determined the pulsotype of and identified the ESBL in all non-redundant strains isolated between 2009 and 2010. RESULTS: The incidence for 1000 hospitalization days increased from 0.00 to 0.44 for ESBL-producing E. coli, from 0.012 to 0.24 for ESBL-producing K. pneumoniae, from 1999 to 2010. Fifty-three different clones of E. coli were identified among the 61 genotyped isolates. The 28 K. pneumoniae isolates genotyped clustered into 11 different clones, among which one major epidemic clone that included 18 isolates. Respectively 66 and 75% of E. coli and K. pneumoniae isolates produced a CTX-M group 1 ESBL. CONCLUSION: The hospital seems to play a different role in the amplification of ESBL according to the producing species (K. pneumoniae or E. coli). ESBL-producing E. coli seem to have a limited cross-transmission within the hospital and seem to be added to non-producers. Conversely, ESBL-producing K. pneumoniae seem to be cross-transmitted within the hospital and to replace non-producers.