Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae have rapidly spread worldwide and pose a serious threat for health care-associated (HA) infection. We conducted molecular detection and characterization of ESBL-related bla genes, including bla(TEM), bla(SHV), bla(CTX-M), bla(VEB), bla(OXA), bla(PER), and bla(GES), among 362 isolates of ESBL-producing E. coli (n = 235) and ESBL-producing K. pneumoniae (n = 127) collected from patients who met the definition of HA infection at two major university hospitals in Thailand from December 2004 to May 2005. The prevalence of ESBL-producing E. coli and ESBL-producing K. pneumoniae, patient demographics and the susceptibilities of these bacteria to various antimicrobial agents were described. A total of 87.3% of isolates carried several bla genes. The prevalence of bla(CTX-M) was strikingly high: 99.6% for ESBL-producing E. coli (CTX-M-14, -15, -27, -40, and -55) and 99.2% for ESBL-producing K. pneumoniae (CTX-M-3, -14, -15, -27, and -55). ISEcp1 was found in the upstream region of bla(CTX-M) in most isolates. Up to 77.0% and 71.7% of ESBL-producing E. coli and ESBL-producing K. pneumoniae, respectively, carried bla(TEM); all of them encoded TEM-1. ESBL-producing K. pneumoniae carried bla(SHV) at 87.4% (SHV-1, -2a, -11, -12, -27, -71, and -75) but only at 3.8% for ESBL-producing E. coli (SHV-11 and -12). bla genes encoding VEB-1 and OXA-10 were found in both ESBL-producing E. coli (8.5% and 8.1%, respectively) and ESBL-producing K. pneumoniae (10.2% and 11.8%, respectively). None of the isolates were positive for bla(PER) and bla(GES). Pulsed-field gel electrophoresis analysis demonstrated that there was no major clonal relationship among these ESBL producers. This is the first study to report CTX-M-3, CTX-M-27, CTX-M-40, SHV-27, SHV-71, and SHV-75 in Thailand and to show that CTX-M ESBL is highly endemic in the country.