Listeria monocytogenes is a pathogen responsible for listeriosis, a foodborne disease with high mortality rates (20–30%). It mainly affects the elderly, pregnant women, and immunocompromised people. Although not pathogenic, the isolation and identification of Listeria innocua are critical since they can indicate L. monocytogenes’ presence as they are closely related and widely distributed in the environment and food processing plants. The objective of this study was to evaluate the effectiveness of the automated methods VITEK® 2 and MALDI-TOF/MS in identifying 94 strains of the genus Listeria with atypical identification profile. The resulting identification by Polymerase Chain Reaction (PCR), using specific primers for the most common species of Listeria, was considered the correct identification and presented a total of 31 strains identified as Listeria innocua (LI), 54 as L. monocytogenes (LM), 8 as Listeria welshimeri (LW) and 1 as Listeria grayi (LG). The VITEK® 2 automated system correctly identified, on average, 79% of the LI strains, 16% of the LM strains, and 88.0% of the LW strains. In the analysis by MALDI-TOF/MS, on average, 73% of LM strains were correctly identified, few LW strains were correctly identified, and all LI strains were incorrectly identified. Both VITEK® 2 and MALDI-TOF/ MS correctly identified the LG strain in both analyzes. The results demonstrate that automated methodologies could not discriminate atypical strains of the Listeria genus and point to the need for the use of complementary tests, such as PCR and chromogenic media, for the correct identification of these strains.2025-02