Objective To understand the multidrug-resistant organism (MDRO) infection in different specialized intensive care units (ICUs) for 5 consecutive years, and provide reference basis for the rational use of antimicrobial agents in different specialized ICUs. Methods Data of MDROs isolated from inpatients in 9 different specialized ICUs in a tertiary first-class hospital between January 2016 and December 2020 were retrospectively studied, isolation rate, discovery rate, infection site and antimicrobial resistance rate of MDROs in different specia-lized ICUs were analyzed. Results From 2016 to 2020, 30 030 inpatients in different specialized ICUs were monitored, 2 744 strains of MDROs were isolated, including 1 235 strains of healthcare-associated infection (HAI) pathogens, there was significant difference in the isolation rates of different kinds of MDROs during 5 years (P < 0.05); there were significant differences in the isolation rates of carbapenem-resistant Pseudomonas aeruginosa (CRPA) and carbape-nem-resistant Klebsiella pneumoniae (CRKP) in different years (all P < 0.05), and there was an annual upward trend. Discovery rate of MDRO infection in 5 years was 3.86%, and case infection rate was 4.11%; there were significant difference in the case incidence of MDRO infection in different specialized ICUs in different years and 5 years (all P < 0.05); there was no significant difference in the case incidence of MDRO infection in all ICUs in different years (P>0.05). The top 3 sites of MDRO infection were lower respiratory tract (578 cases), blood (240 cases) and urinary tract (170 cases). Resistance rates of CRAB isolated from general ICU, cardiovascular surgery ICU, neurosurgery ICU and emergency ICU to gentamicin and ciprofloxacin were all significantly different (all P < 0.05), resistance rates of CRPA to gentamicin were significantly different (all P < 0.05). Resistance rates of CRAB to ce-foperazone/sulbactam and CRPA to piperacillin, piperacillin/tazobactam as well as cefo-perazone/sulbactam were all significantly different (all P < 0.05). Conclusion During the process of clinical diagnosis and treatment, infection prevention and control measures and rational use of antimicrobial agents should be guided according to the characte-ristics of MDRO infection in different specialized ICUs.