Objective To investigate the clinical drug resistance of carbapenem-resistant Gram-negative bacilli and molecular characteristics of drug resistance gene blaKPC. Methods Clinically isolated carbapenem-resistant Gram-negative bacilli in a hospital from January 2017 to December 2018 were analyzed. Antimicrobial susceptibility testing data were statistically analyzed by WHONET 5.6 software, carbapenem-resistant genes blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48 were detected by polymerase chain reaction (PCR), PCR positive products were performed DNA sequence to analyze the molecular structure of drug resistance genes. Results A total of 510 strains of carbapenem-resistant Gram-negative bacilli were collected, including 420 strains of carbapenem-resistant Enterobacteriaceae (CRE) and 90 strains of carbapenem-resistant non-Enterobacteriaceae. Strains were mainly from intensive care unit (ICU), departments of neurosurgery and respiratory, accounting for 60.8%, 11.8% and 5.3% respectively; specimens were from sputum, purulent secretion, venous blood, and sterile midstream urine, accounting for 66.9%, 8.8%, 8.2% and 6.5% respectively. Carbapenem-resistant Gram-negative bacilli were highly resistant to commonly used antimicrobial agents. PCR results showed that among 420 strains of CRE, positive rates of blaKPC, blaNDM, and blaIMP were 54.3% (228/420), 1.2% (5/420), and 1.4% (6/420) respectively. blaVIM and blaOXA-48 genes were not found, Klebsiella pneumoniae, Enterobacter aerogenes, and Escherichia coli accounted for 83.8%, 11.8%, and 2.6% of blaKPC-carrying CRE respectively; blaKPC was also detected in other rare strains. blaKPC was detected in only 2 strains of Acinetobacter baumannii in non-Enterobacteriaceae. DNA sequencing showed that among 174 strains carrying blaKPC, 173 strains were found blaKPC-2 and 1 strain was found blaKPC-1. Conclusion CRE is the main Gram-negative bacteria resistant to carbapenems in this area, dominantly Klebsiella pneumoniae carrying blaKPC-2, and other strains are also found. It is suggested that the monitoring and prevention of carbapenem-resistant Klebsiella pneumoniae should be strengthened to prevent and control the spread of blaKPC.