Abstract: Objective To investigate the association between molecular characteristics and antibiotics resistance in clinical isolates of Enterobacter cloacae. Methods A total of 72 isolates of E. cloacae from a hospital were typed according to hsp60 gene sequence and peptide mass fingerprinting. The susceptibilities of the isolates to 14 antibiotics were tested with agar dilution method. Twenty antibiotic resistance related genes were analyzed by using PCR and sequenced. Results Based on hsp60 genotype and peptide mass fingerprinting analysis,72 E. cloacae strains could be divided into 2 clades and 9 gene clusters, clade 1(gene cluster:Ⅲ,Ⅵ and Ⅷ)strains were predominant in all the clinical isolates(50 strains,69.4%). Antibiotic susceptibility test showed that the isolates were resistant to cefazolin,cefuroxime,ceftriaxone,ceftazidime,ampicillin/sulbactam;but sensitive to cefepime,piperacillin/tazobactam,aminoglycoside and quinolone. Eight antibiotics resistance genes (mir-act,dha,shv,tem. ctx-M,qnrA,qnrB and qnrS) were detected. Conclusion Predominant genotype was detected in the clinical isolates of E. cloacae with two subtyping methods. There were significance differences in clinical distribution and antibiotic resistance among the E. cloacae strains with different genotypes.