Objective To study the molecular epidemiological characteristics of drug resistance of carbapenem-resistant Enterobacteriaceae (CRE) in a hospital, and provide evidence for the prevention and control of CRE. Methods CRE stored in the bacterial laboratory of a hospital from 2013 to 2017 were collected and performed multilocus sequence typing (MLST), antimicrobial susceptibility testing as well as whole-genome sequencing, carbapenem resistance genes carried in some CRE strains were conducted gene environment analysis. Results A total of 62 strains of CRE were collected and 51 were successfully revived, including 30 strains of carbapenem-resistant Klebsiella pneumoniae(CRKP), 9 strains of carbapenem-resistant Escherichia coli (CREC),6 strains of carbapenem-resistant Enterobacter cloacae (CRECL), and 6 strains of other CRE. CRKP MLST mainly included 3 ST147 strains and 2 ST11 strains; CREC MLST mainly included 3 ST167 strains; CRECL MLST mainly included 3 ST93 strains and 2 ST88 strains. 51 strains of CRE showed the highest resistance to ampicillin and cefotaxime, all were 100%. Distribution of carbapenem resistance genes was follows:1, 14, 18, 22, 2,10, 10, 2, and 2 strains carried bla_KPC-2, bla_IMP-4, bla_NDM-1, bla_NDM-5, bla_NDM-9, bla_OXA-1, bla_OXA-10, bla_OXA-23, and bla_OXA-66 respectively. Genetic environment of bla_NDM-1, bla_NDM-5, bla_NDM-9, and bla_IMP-4 in different strains was analyzed, it was found that the genetic environment of several drug-resistant genes was similar to the previously reported genetic environment, there was no significant difference among strains. Conclusion Drug resistance genes can be stable in different CRE strains through horizontal transmission, which poses a threat to the prevention and control of healthcare-associated infection.