Abstract: OBJECTIVE To investigate the antibacterial drug usage and the detection of major multidrug-resistant bacteria in 92 secondary and higher-level hospitals in Shanghai, compare the difference between secondary and tertiary hospitals, explore the correlation between the antibacterial drug usage and the detection of multidrug-resistant bacteria, so as to provide a basis for precise prevention and control. METHODS A retrospective survey was conducted in Feb. 2023 through online reporting forms, covering the antibacterial drug usage and the detection of drug-resistant bacteria in patients from 92 hospitals in Shanghai from Jan. 1 to Dec. 31, 2022. Data were analyzed with SPSS 23.0 statistical software. RESULTS A total of 92 hospitals were included, with 50 secondary hospitals and 42 tertiary hospitals. The usage rate of antibacterial drugs in secondary hospitals was higher than that in tertiary hospitals (46.06% vs. 38.86%, χ²=10 855.056, P＜0.001). The detection rates of methicillin-resistant Staphylococcus aureus (53.00% vs. 45.29%), vancomycin-resistant Enterococcus (0.68% vs. 0.09%) and carbapenem-resistant Escherichia coli (3.29% vs. 2.84%) were all higher in secondary hospitals than in tertiary hospitals (P＜0.05). Conversely, carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Klebsiella pneumoniae were more prevalent in tertiary hospitals than in secondary hospitals (P＜0.05). The increase in antibacterial drug usage was associated with an elevated detection rate of vancomycin-resistant Enterococcus (P＜0.05). CONCLUSIONS Differences in hospital levels affect drug resistance spectrum characteristics. Strengthening the rational use of antibacterial drugs is key to controlling multidrug-resistant bacteria. Precise antibacterial drug management and infection prevention and control measures should be developed and implemented in accordance with the characteristics of hospitals of different levels.