Abstract: Objective：To investigate the genotype of carbapenem-non-susceptible Enterobacteriaceae isolated from clinical specimens in our hospital. Methods：A total of 110 clinical isolates of carbapenem-non-susceptible Enterobacteriaceae were isolated from clinical specimens in our hospital during January 2010 to June 2013. The susceptibility of isolates to carbapenems was determined by the disk diffusion method. The extended spectrum β-lactamases (ESBLs) and the carbapenemase phenotypes of isolates were detected by the phenotypic confirmatory test and the modified Hodge test (MHT), respectively. Further, the genotypes of β-lactamases were determined by polymerase chain reaction (PCR), DNA sequencing and BLAST aligning. Results：Of 110 isolates of carbapenem-non-susceptible Enterobacteriaceae, 77 (70.0%) were positive for carbapenemase. PCR and DNA sequencing analysis showed that 31 isolates of E. coli, 1 E. aerogenes and 1 S. marcescens carried with bla_KPC-2 gene, 5 isolates of E. cloacae with bla_IMP-26 and 1 E. aerogenes with bla_VIM-2. In addition, 36 (32.7%) of isolates produced ESBLs, and 3 genotypes, such as bla_CTX-15, bla_SHV-12 and bla_CTX-3, were detected. Conclusion: bla_KPC-2 was the major genotype of carbapenemase for the clinical isolates of carbapenem-non-susceptible Enterobacteriaceae in our hospital, followed by bla_IMP-26 and bla_VIM-2.