Abstract: OBJECTIVE To investigate the resistance profile of clinical isolates to commonly used antibiotics collected from a newly established hospital in Shanghai, so as to provide reasonable medication basis for clinicians in our hospital METHODS All clinical isolates from Jan. 2016 to Dec. 2016 were selected, and the resistance was monitored. RESULTS A total of 992 clinical isolates were collected in 2016, of which 282 strains were gram-positive bacteria and 710 strains were gram-negative bacteria, accounting for 28.43% and 71.57%, respectively. The detection rates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MRCNS) were 50.49%(52/103) and 58.33%(21/36) respectively. The resistant rates of MRSA and MRCNS to most antimicrobial agents were much higher than those of MSSA and MSCNS (P<0.05). MRSA strains were susceptible to trimethoprim-sulfamethoxazole and rifampin, and MRCNS strains were susceptible to rifampin and minocyline. All Staphylococcus strains were susceptible to vancomycin and linezolid. In Enterococcus spp., the resistant rates of E. faecalis strains to most of the antibiotics tested were much lower than those of E. faecium, and strains of both species were susceptible to vancomycin. The prevalences of ESBLs-producing strains were 50.22%(113/225) in E. coli and 32.40%(58/179) in Klebsiella spp. isolates on average. ESBLs-producing Enterobacteriaceae strains were more resistant than non-ESBLs-producing strains in terms of antibiotic resistance rates. The strains of Enterobacteriaceae were still highly susceptible to carbapenems but K. pneumoniae. About 76.41% of Acinetobacter spp. strains were resistant to imipenem and meropenem, respectively. CONCLUSION The isolation rate of drug resistant strains increased, suggesting that the widespread epidemic tendency of extensively drug-resistant bacteria in some areas, therefore, it is necessary to rationally use antibiotics, strengthen the monitoring of bacterial resistance, and actively control nosocomial infection.