Mutations can increase the ceftazidimase activity of CTX-M-3 β-lactamase, as seen with its widespread variant CTX-M-15. This study compared the frequencies of emerging ceftazidime resistance in isogenic wild-type and hyper-mutable mutS CTX-M-3-producing Escherichia coli strains, and sequenced the mutant blaCTX-M alleles selected. Ceftazidime resistance emerged more readily in the hyper-mutable background than in the wild-type strain. All selected CTX-M mutants, in both the wild-type and the mutS derivatives, had single amino-acid changes at position 167, including a novel Pro167Gln substitution. These data emphasise the potential for further diversification of CTX-M enzymes.