Background: The aim of this study was to determine the prevalence of K. pneumoniae encoded bla CMY-2 gene, isolated from clinical specimens. Methods: In this Analytic-descriptive cross-sectional study, 144 isolates of Klebsiella spp. were collected from the clinical specimens such as wounds (7cases), supra pubic (1 case), blood (8 cases), septum (2 cases), catheter tips (3 cases), urine (106 cases), CSF (1 case), skin lesion (1 case), tracheal (9 cases), anal (2 cases), throat (1 cases) and eye culture (3 case) from Rasht hospitals from February to August 2013. After identification of isolates by biochemical methods, the antibiotic susceptibility test (Kirby-Bauer method) was done according to CLSI guideline against 20 antibiotics. The combined disk method (Double disk) was then carried out for detection of ESBLs of Klebsiella spp. Among ESBL producers of Klebsiella spp. blaCMY-2 was detected by PCR using specific primers. Then, PCR products were subjected to electrophoresis on 1.5 % agarose gel. Finally, PCR products were confirmed by sequencing.Results: Among the Klebsiella 144 clinical isolates, 57 (39.6%) isolates were ESBL producers. The most prevalent ESBL producers were isolated from urine sample (33.57). The most resistance in Klebsiella spp. were belong to Oxacillin and Amoxicillin (98.1% and 97.2%, respectively) and Imipenem was the most effective antibiotic (95.3%) against all isolates. Among 57 ESBL producers of Klebsiella spp. only 12 cases (21.01%) were contained blaCMY-2 gene. This is the first report of blaCMY-2 gene found in Klebsiella spp. in Iran.Conclusion: Due to high frequency of ESBL producing Klebsiella spp. isolated from clinical specimens, antibiogram should be conducted to choose the best antibiotic against Klebsiella spp and empirical treatment should be avoided to reduce antibiotic resistance development.