ABSTRACT Aim: To characterize temporal shifts in extended-spectrum β-lactamases (ESBLs) and clones of clinical Escherichia coli isolates. Materials & methods: All ESBL-producing E. coli isolates from a Portuguese hospital (n = 112; June 2006–June 2007 and January–December 2010) were characterized by identification of phylogenetic groups, ESBL-types and virulence genes, XbaI-PFGE and MLST. Results: We observed a substantial increase in widespread E. coli clones from phylogroups A, B1 and D (e.g., ST10, ST23, ST117, ST155, ST648) producing mainly CTX-M-1, -14, -32 or SHV-12, along with a decrease in the proportion of the predominant CTX-M-15-producing B2-ST131 clone. Conclusion: The amplification of diverse CTX-M-producing A, B1 and D clonal complexes, which have been long identified in Portuguese nonclinical settings, unveils a role for these reservoirs in the landscape of ESBL-producing E. coli in the clinical setting.