Abstract: OBJECTIVE To analyze the transmission routes of multidrug-resistant Acinetobacter baumannii (MDR-AB) in the intensive care unit (ICU) by integrating epidemiological and molecular biological methods. METHODS The sputum specimens and related information were collected from the patients with lower respiratory tract infections of MDR-AB who were treated in the first department of critical care medicine, second department of critical care medicine, and ICU of neurosurgery department of the First Affiliated Hospital of Shihezi University from Jan. 2020 to Dec. 2020. The samples were collected from surroundings of the patients and hands of health care workers, species of pathogens were identified, multilocus sequence typing (MLST) and cluster analysis were performed. The major strains prevalent in the ICUs were analyzed, and the transmission routes of homologous MDR-AB strains were inferred based on the homology between the patient strains and strains from the surrounding environment and healthcare workers' hands and the epidemiological evidence such as the relationship between bed location and genotype distribution, as well as the timing of MDR-AB infection. RESULTS Totally 174 strains of MDR-AB were isolated from the patients and their surrounding environments, including 33 strains from patient samples and 141 strains from the surrounding environment and healthcare workers' hands. Totally 18 ST types were identified, with ST540 being the predominant epidemic strain. In the first department of critical care medicine, MDR-AB primarily showed a sporadic pattern throughout the year, and the transmission mainly occurred through shared medical equipment in the ward. In the ICU of neurosurgery department, an outbreak of MDR-AB health care-associated infection in December was attributed to ST540, with the transmission occurring through cross-contamination among the patients, environment, and healthcare workers' hands. CONCLUSIONS The predominant epidemic strain of MDR-AB is ST540 in the ICU, and the transmission primarily occurs through cross-contamination among the patients, the environment, and the healthcare workers' hands.