Objective: The objective of this study was to evaluate the performance of five commonly used automated antimicrobial susceptibility testing (AST) systems in China (Vitek 2, Phoenix, Microscan, TDR, and DL). Materials and methods: Two “unknown” isolates, S1 (ESBL-producing Escherichia coli) and S2 (KPC-producing Klebsiella pneumoniae), were sent to 886 hospitals in China for identification and AST. Using broth microdilution method (BMD) as gold standard, minimum inhibitory concentrations (MICs) were determined. Results: Most hospitals (392, 46.1%) used Vitek 2, followed by 16% each for Phoenix, Microscan, and DL systems, and 5.9% (50) used TDR system. MICs of 22 antimicrobials were evaluated for two study isolates plus three ATCC strains. Individual susceptibility results for three ATCC strains (n=1581) were submitted by 780 (91.2%) hospitals. For each AST system, 8.7% (6/69) to 13.0% (33/253) reported MICs outside the expected range for several drugs. For the two study isolates, TDR and DL systems performed the worst in MIC determination and susceptibility categorization of cefazolin and cefepime, while the Microscan system had difficulties in susceptibility categorization for aztreonam and ertapenem. Categorical agreements were >90% for most antimicrobials tested for both the isolates, among which, using BMD, no essential agreements were noted for ampicillin, piperacillin, cefazolin, cefuroxime, ceftriaxone, and trimethoprim/sulfamethoxazole. All AST systems except Vitek 2 showed unacceptable VMEs for cefazolin (S1 and S2) and major errors for ceftazidime, cefepime, and aztreonam (isolate S1), while Vitek 2 showed a high VME rate for cefepime (10.0%) and meropenem (6.2%) for S2. Conclusion: None of the five automated systems met the criteria for acceptable AST performance, but Vitek 2 provided a relatively accurate and conservative performance for most of the antimicrobials.