Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli has spread rapidly worldwide and poses a serious threat to human and animal health. This study collected 51 non-replicate E. coli isolates from 14 different chicken farms in Henan Province in China from December 2007 to August 2008. The prevalence of ESBL-producing E. coli, molecular characterization of the ESBL-related bla genes, including bla(TEM), bla(SHV) and bla(CTX-M), and the susceptibilities of these bacteria to various antimicrobial agents were determined. Thirty-one of the 51 isolates were positive for an ESBL phenotype and 29 of these isolates carried one or more bla genes. Twenty-two isolates harboured bla(TEM) genes and 15 isolates carried bla(CTX-M) genes (one CTX-M-14, three CTX-M-24 and 11 CTX-M-65). One isolate carried bla(TEM)(-57); the remaining bla(TEM) isolates carried bla(TEM-1) with one silent nucleotide base variation (T18C). We believe that this is the first study to report TEM-57 in E. coli isolates. All isolates harbouring bla(CTX-M-24) and bla(CTX-M-14) and five of the bla(CTX-M-65) isolates also harboured the bla(TEM-1) gene. To our knowledge, this study is the first to describe detection of CTX-M-65-producing E. coli isolated from chickens. None of the isolates contained the bla(SHV) gene. Conjugation experiments demonstrated that bla(CTX-M) and bla(TEM) genes could be transferred to E. coli DH5 alpha. The results indicate that ESBL frequency has reached an alarming level in chicken isolates in China, with TEM-1 and CTX-M-65 enzymes being the two predominant beta-lactamases detected.