Objective To investigate the homology of the multidrug-resistant organisms (MDROs) isolated from patients and environment of intensive care unit (ICU) in a hospital, and provide evidence for clinical prevention and control of MDRO infections. Methods MDROs isolated from patients and environment of ICU in a tertiary first-class hospital from November 17 to December 17, 2021 were collected. Antimicrobial susceptibility test, pulsed-field gel electrophoresis (PFGE) and cluster analysis were conducted on the isolated strains. Results A total of 22 MDROs were collected, including 6 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains, 11 multidrug-resistant Acinetobacter baumannii (MDR-AB) strains and 5 methicillin-resistant Staphylococcus aureus (MRSA) strains. 11 strains each were isolated from clinical patients and environment. Some of the environmental strains were highly homologous to the clinical strains. MDROs were isolated from ICU environment such as the surfaces of objects in the staff's living area, microwave oven buttons, switches, etc. CRKP and MDR-AB were isolated even after disinfection of patient bedside tables and sinks. MRSA identical to that from the stethoscope used by patient was isolated from nurses' hands after hand washing. CRKP was resistant to all commonly used antimicrobial agents (except ceftazidime/avibactam). MDR-AB clinical strains showed a resistance rate of 40.0% to minocycline, and resis-tance rates ≥ 60% to other antimicrobial agents; while environmental strains of MDR-AB had a lower resistance rate than the clinical strains. Conclusion Clinical MDRO strains from ICU are highly homologous to the environmental strains, suggesting the possibility of MDRO transmission and cross-infection through the hospital environment. Therefore, it is necessary to strengthen the implementation of environmental cleaning and disinfection measures, improve hand hygiene compliance, and cut off the transmission of pathogenic bacteria in hospital to reduce the incidence of MDROs.