The aim of this study was to characterize by a multi-level approach extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates other than E. coli from Portuguese hospitals. Eighty-eight ESBL-producing clinical isolates (69 Klebsiella pneumoniae, 13 Enterobacter cloacae complex, 3 Klebsiella oxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis and 1 Serratia marcescens) recovered from hospitals located in the North (A) or Centre (B, C) regions during two time periods (2006-7 and 2010) were analyzed. Standard methods were used for bacterial identification, antibiotic susceptibility testing, ESBL characterization, clonal (PFGE, MLST) and plasmid (S1-PFGE, I-CeuI-PFGE, replicon typing, hybridization) analysis. Isolates produced mostly CTX-M-15 (47%) or SHV-12 (30%), and less frequently other SHV- (15%; SHV-2, -5, -28, -55, -106) or TEM- (9%; TEM-10, -24, -199)-types, with marked local and temporal variations. The increase of CTX-M-15 and diverse SHV ESBL-types observed in Hospital A was associated with the amplification of multidrug-resistant (MDR) K. pneumoniae epidemic clones (ST15, ST147, ST336). SHV-12 and TEM-type ESBLs were mostly identified in diverse isolates of different Enterobacteriaceae species in Hospitals B and C in 2006-7. Particular plasmid types were linked to blaCTX-M-15 (IncR or non-typeable plasmids), blaSHV-12 (IncR or IncHI2), blaSHV-28/-55/-106 (IncFIIK1 or IncFIIK5), blaTEM-10 (IncL/M) or blaTEM-24 (IncA/C), mostly in epidemic clones. In our country, the amplification of CTX-M-15 and diverse SHV-type ESBL among non-E. coli Enterobacteriaceae is linked to international MDR K. pneumoniae clones (ST15, ST147, ST336) and plasmid types (IncR, IncFIIK). Furthermore, we highlight the potential of IncFIIK plasmids (here firstly associated with blaSHV-2/-28/-55/-106) to disseminate as antibiotic resistance plasmids.