The production of extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae is the most prevalent resistance mechanism to third-generation cephalosporins. The aim of this study was to identify the ESBLs produced in Escherichia coli and Klebsiella pneumoniae clinical isolates from two hospitals of the Colombian Caribbean Region. A total of 30 clinical isolates of K. pneumoniae (21) and E. coli (9) ESBL-producers were collected in two hospitals from January, 2001 to June, 2003. Isoelectric point values were indicative of SHV-, and CTX-M-type beta-lactamases. PCR amplification and sequencing of SHV genes revealed that SHV-12 was the most prevalent ESBL followed by SHV-5, SHV-2a, the novel SHV-86 and CTX-M-12. There was a geographic distribution of two particular PFGE subtypes in these two distant hospitals. Clonal and horizontal dissemination of resistance was observed.