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Abstract Background Commercial diagnostics are commonly used to identify gram-positive bacteria. Errors have been reported mostly at the species level. We have found certain phenotypic criteria used in API systems which significantly misidentify Leuconostoc, an emerging human pathogen, at the genus level. We also attempt to find practical, conventional phenotypic assays for accurate identification of this group of bacteria. Methods Clinical isolates of catalase-negative, gram-positive coccoid or coccobacillary bacteria with non-β hemolysis in our institute during 1997–2004 were subject to an identification aid by API 20 STREP, following the instruction manual, as an aid to conventional phenotypic tests. Those identified as Leuconostoc by API 20 STREP were re-examined by the same kit and also by API 50 CHL according to the instruction manuals, by our Leuconostoc conventional phenotypic assays, by Leuconostoc- and Lactobacillus-specific PCR's, and, where possible, by 16S rDNA sequence analysis. In addition, catalase-negative gram-positive isolates during 2005–2006 which were resistant to vancomycin at high levels were also evaluated by the same phenotypic and genotypic assays. Results Out of several thousands of clinical gram-positive isolates, 26 catalase negative gram-positive isolates initially identified as Leuconostoc by API 20 STREP and 7 vancomycin-resistant gram-positive catalase-negative bacteria entered the study. 11 out of the 26 isolates and all the 7 isolates were identified as Leuconostoc by API 20 STREP. Only 5 isolates, however, were confirmed by both genotypic and all defined conventional phenotypic criteria. API 50 CHL also failed to reliably provide accurate identification of Leuconostoc. We have identified key problem tests in API 20 STREP leading to misidentification of the bacteria. A simple, conventional set of phenotypic tests for Leuconostoc identification is proposed. Conclusion The current API systems cannot accurately identify Leuconostoc. Identification of vancomycin-resistant, catalase-negative gram-positive bacteria should be performed by a few practical phenotypic assays, with assistance of genotypic assays where available.