This study reports phenotypic and molecular characterization of a novel CTX-M beta-lactamase carried by two Klebsiella pneumoniae isolates collected from two hospitals in China. Conjugation experiment, Southern hybridization, susceptibility testing, isoelectric focusing, PCR, and sequencing techniques as well as clone, expression, purification and kinetics were carried out to describe the characterization of the novel CTX-M-type enzyme. The analyses of plasmid profiling and pulsed-field gel electrophoresis of the novel enzyme were performed to investigate epidemiology. The PCR products had 967 nucleotides and a novel CTX-M enzyme with a pI of 8.5 was implicated in this resistance: CTX-M-72. Two strains exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. The amino acid sequence of the CTX-M-72 beta-lactamase differed from that of the CTX-M-3 beta-lactamase by the Arg-->Gly change at position 164. The novel enzyme was susceptible to ceftazidime, the same response being observed for other CTX-M enzymes. The substrates of the beta-lactamase were also characterized. Furthermore, two resistant genes of clinical strains were closely related. The emergence of a novel CTX-M-type extended-spectrum beta-lactamase was rarely described in other areas. This study illustrated the importance of molecular surveillance in tracking CTX-M-producing strains in large teaching hospitals, suggested the horizontal transfer of plasmid-borne bla(CTX-M) genes contributed to the dissemination of CTX-M enzymes in hospital environments, and emphasized the need for epidemiological monitoring.