Objective To investigate the distribution and antimicrobial resistance of common pathogens from clinical specimens nationwide in 2020-2024, provide scientific basis for clinical rational application of antimicrobial agents, as well as for the government to timely grasp the antimicrobial resistance situation nationwide, formulate and evaluate clinical application management policies for antimicrobial agents. Methods According to the technical scheme of China Antimicrobial Resistance Surveillance System (CARSS), antimicrobial resistance of all bacterial isolates reported by CARSS in 2020-2024 were analyzed with WHONET 5.6 software. Results The top 5 isolated Gram-positive bacteria from tertiary hospitals nationwide were Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Staphylococcus epidermidis, and Streptococcus pneumoniae. The top 5 isolated Gram-positive bacteria from secondary hospitals were Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Staphylococcus epidermidis, and Enterococcus faecium. Isolation rate of Streptococcus pneumoniae from secondary hospitals was higher than that from tertiary hospitals. The percentage of top 5 isolated Gram-negative bacteria in 2020-2024 were Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter cloacae. In 2024, the isolation rate of Haemophilus influenzae in tertiary and secondary hospitals ranked the fifth. Detection rates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase negative Staphylococcus (MRCNS) have decreased in tertiary hospitals. Detection rates of vancomycin-resistant Enterococcus faecium (VREM) increased in tertiary and secondary hospitals. The detection rates of carba-penem-resistant Pseudomonas aeruginosa (CRPAE) decreased in hospitals of different levels, while those of carba-penem-resistant Acinetobacter baumannii (CRABA) showed no significant changes; in the same period, the detection rates of both pathogens in tertiary hospitals were higher than those in secondary hospitals. Detection rates of cefotaxime-resistant Escherichia coli and Klebsiella pneumoniae have declined, but both were higher in tertiary hospitals than in secondary hospitals during the same period. Detection rates of imipenem- and meropenem-resistant Escherichia coli didn’t change much and both were lower than 1.8%; Resistance rates of Klebsiella pneumoniae to imipenem and meropenem decreased, with tertiary hospitals having a significantly higher resistance rate to imipenem and meropenem than secondary hospitals. Resistance rates of Haemophilus influenzae to most antimicrobial agents increased, and that from tertiary hospitals were higher than from secondary hospitals during the same period. Conclusion The detection rates of MRSA and MRCNS have decreased in tertiary hospitals, whereas the changes in secondary hospitals remain insignificant. The detection rates of VREM have increased in both tertiary and secondary hospitals. In tertiary hospitals, the detection rate of CRKPN (carbapenem-resistant Klebsiella pneumoniae) remains at a high level, and is notably higher than that in secondary hospitals. Acinetobacter baumannii exhibits severe resistance to various antimicrobial agents in hospitals of different levels, and the drug resistance rate of Haemophilus influenzae to multiple antimicrobial agents has increased. The management of rational use of antimicrobial agents as well as prevention and control of healthcare-associated infection should continue to be strengthened, and surveillance of antimicrobial resistance should be conducted well.