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Objective:To identify three common genes (blaTEM, blaSHV and blaCTX-M) responsible for ESBL production in Klebsiella pneumoniae (K. pneumoniae) isolated from Intensive Care Units of Namazi Hospital, Shiraz, Iran. Methods:A total of 60 non-repetitive nosocomial isolates from 60 patients were selected during 2009-2010. The phenotypic identification of ESBL production was confirmed by Double Disk Synergy Test (DDST) according to CLSI guidelines. The ESBL’s genotype was then analyzed by multiplex PCR of blaTEM, blaSHV and blaCTX-M genes and DNA sequencing. Results: The primary susceptibility tests of K. pneumoniae showed that among 10 examined antibiotics, the most resistant and susceptible antibiotics identified in this study were ampicillin and imipenem, respectively. The phenotypic determination of ESBL by DDST showed that 60%(n=36) of isolates produced ESBL. Multiplex PCR of genes among K. pneumoniae isolates showed that 39% (n=18) of them have TEM, 39% (n=18) of them have both CTX-M and TEM and 13% (n=8) of them have TEM, SHV, CTX-M. Conclusions:Our findings reveal the high prevalence (60%) of ESBL producing K. pneumoniae from ICU patients along with a new patt of blaTEM distribution differ from other countries.