BACKGROUND: Several automated and nonautomated systems have been developed and are commercially available for the identification of gram-negative bacilli. EASY 24E+ kit was recently developed as Korean kit for identification of gram-negative bacilli. So we evaluated the accuracy and clinical utility of EASY 24E+ compared with API 20E and VITEK GNI+. METHODS: The 221 clinical isolates of Enterobacteriaceae, including 17 C. freundii, 20 E. cloacae, 31 E. coli, 6 E. aerogenes, 29 K. pneumoniae, 3 K. oxytoca, 11 M. morganii, 13 P. mirabilis, 16 Salmonella spp., 20 S. marcescens, 9 Shigella spp., 22 S. sonnei, 16 S. typhi, 8 Y. pseudotuberculosis and 10 control strains were identified by API 20E, EASY 24E+, and VITEK GNI+. Discrepant strains were performed repeat identifications and we evaluated overall accuracy. RESULTS: All of control strains were correctly identified by three systems. The overall correct results at species level and at the genus level for 221 clinical isolates, were 96.8% and 99.1% by the VITEK GNI+, 97.7% and 97.7% by the EASY 24+ and 99.1% and 100% by the API 20E. All of Salmonella spp., S. typhi and Shigella spp. were correctly identified by all three systems and the discrepant identifications of species were 2 Y. pseudotuberculosis, 3 K. pneumoniae and 2 K. oxytoca by VITEK GNI+, 4 C. freundii and 1 P. mirabilis by EASY 24+, and 2 S. marcescens by API 20E. CONCLUSIONS: All three identification systems are accurate methods for the identification of Enterobacteriaceae, and EASY 24+ is comparable with API 20E and VITEK GNI+.