This study was conducted to detect the genes encoding extended-spectrum beta-lactamases (ESBLs) and determine the epidemiological relatedness of 69 Escherichia coli and 33 Klebsiella pneumoniae isolates collected from a regional hospital in central Taiwan, mostly from inpatients (E. coli 87.0%; K. pneumoniae 88.0%). The phenotypes of these isolates were examined according to the combination disc method recommended by the Clinical and Laboratory Standards Institute. Most of the ESBL-producing E. coli and K. pneumoniae isolates (98.6% and 97%, respectively) could be detected using cefotaxime discs with and without clavulanate. Genotyping was performed by PCR with type-specific primers. CTX-M-14 type (53.6%) was the most prevalent ESBL among E. coli isolates while SHV type (57.6%) was the most dominant among K. pneumoniae isolates. Six E. coli and three K. pneumoniae isolates did not carry genes encoding ESBLs of types TEM, SHV, CTX-M-3, CTX-M-14, CMY-2 and DHA-1. The co-existence of two or more kinds of ESBL in a single isolate was common, occurring in 40.6% and 72.7% of E. coli and K. pneumoniae isolates, respectively. PFGE analysis revealed that ESBL producers isolated in this setting were genetically divergent.