Two clinical strains (Klebsiella pneumoniae 516 and K. pneumoniae 1335) collected in September 2006 from different hospitals in Anhui Province (China) harboured two novel plasmid-mediated bla(CTX-M) genes, designated bla(CTX-M-80) and bla(CTX-M-81), respectively. Both CTX-M-80 with pI of 9.0 and CTX-M-81 with pI of 8.4 were extended-spectrum beta-lactamases (ESBLs). The results of susceptibility testing demonstrated two enzymes were highly activity against broad spectrum beta-lactams, but the level of resistance was reduced with the addition of beta-lactamase inhibitors. The bla(CTX-M-80) gene was detected on a 110-kb plasmid and the bla(CTX-M-81) gene existed on a 120-kb plasmid. The deduced amino acid sequence of CTX-M-80 differed from that of CTX-M-3 by the substitution Ala-27-->Val, and CTX-M-81 possessed the Lys-->Glu, Lys-->Gln, and Asn-->His changes at respective position 82, 98, and 132 in compassion with CTX-M-14. The enzymatic properties showed CTX-M-80 and CTX-M-81 had higher affinities for penicillin G (lower Km values) than for cephalosporins. The activities of novel enzymes against ceftazidime were undetectable or limited, as indicated by MICs data, the same response being observed for many other CTX-M enzymes. This report was evidence of the diversity of CTX-M-type ESBLs in China.