We evaluated VITEK 2 cards (NGNC and AST-GN10) for the accuracy of identification (ID) and antimicrobial susceptibility testing (AST) of non-glucose-fermenting Gram-negative bacilli (NGF-GNB). In a total of 201 strains, 190 strains (94.5%) were correctly identified, seven strains (3.5%)showed low discrimination, four strains (2.0%) had discrepancies, and no strain remained unidentified.Reference AST of amikacin, aztreonam, cefepime, cefotaxime, ceftazidime, ciprofloxacin, imipenem,levofloxacin, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole was performed by the agar dilution method. Approximately 82.5% of ID and 72.9% of AST were completed within 7 and 14 h,respectively. For NGF-GNB, other than Pseudomonas aeruginosa, Acinetobacter spp., Stenotrophomonas maltophilia, and the Burkholderia cepacia group, essential agreements (EAs) were 93.6-100.0%. Severe disagreements (resistant by the reference method to susceptible by AST-GN10) were observed for amikacin(0.9%), cefepime (1.8%), cefotaxime (1.8%), imipenem (0.9%), and piperacillin-tazobactam (0.9%).One major disagreement (susceptible to resistant) was observed for ceftazidime (0.1%). For P. aeruginosa,EAs were 85.7-100%, with severe disagreements observed for cefepime (4.8%) and piperacillin-tazobactam(4.8%). For Acinetobacter spp., EAs were 86.4-100% without disagreements. The VITEK 2 cards appear to be promising for rapid ID and reliable AST for most species of NGF-GNB.