Background and objectives: Urinary tract infections (UTIs) caused by extended-spectrum beta lactamase (ESBL) -producing bacteria have become a growing problem worldwide. The aim of this study was to investigate the prevalence of ESBL-producing bacteria in urine samples of hospitalized patients in Imam Khomeini hospital of Ardabil over a period of October 2011 to August 2012.Methods: A total of 400 urinary pathogens isolated from urine samples were included in the study. All isolates were identified by routine biochemical methods and antimicrobial susceptibility testing carried out by Kirby-Bauer method. Confirmatory test for production of ESBLs was performed by the combination disk tests. The results were interpreted according to the recommendation of CLSI.Results: Of 400 isolated bacteria, 267 were E.coli, 39 Klebsiella pneumoniae, 17 Klebsiella oxytoca, 16 Enterobacter cloacae, 15 Enterobacter aerogenese, 6 Enterobacter agglomerans, 8 Enterobacter sakazakji, 3 Citrobacter freundii, 2 Citrobacter diversus, 3 Proteus mirabilis, 4 Edvardsiella tarta, 3 Serratia marcesecens and 17 Morganella morganii all of which then were analyzed. ESBL was detected in 36.75% (147) of isolates. One Hundred Fourteen E.coli cases (77.5%), 15 Klebsiella pneumonia (10.2%), 5 Klebsiella oxytoca (3.4%), 4 Enterobacter aerogenese (2.72%), 4 Enterobacter cloacae (2.72%), 1 Citrobacter freundii (0.68%), and 1 Morganella morganii (0.68%) were detected as ESBLs producers, respectively.Conclusion: Based on the results of this study, broad-spectrum beta-lactamase production in bacterial strains isolated from patients with urinary tract infection was very high and almost 40% of all bacterial species isolates were ESBLs producers. Because of the high prevalence of ESBL-producing bacteria in the urinary tract infections in hospitalized patients of our area, we would strongly suggest that the ESBL production should be considered in these patients.