The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for staphylococcal identification is now considered routine in laboratories compared with the conventional phenotypical methods previously used. We verified its microbiological relevance for identifying the main species of coagulase-negative staphylococci (CoNS) by randomly selecting 50 isolates. From 1 January 2007 to 31 August 2008, 12,479 staphylococci were isolated with phenotypic methods, of which 4,594 were identified as Staphylococcus aureus and 7,885 were coagulase negative staphylococci. Using MALDI-TOF MS from 1 January 2011 to 31 August 2012, 14,913 staphylococci were identified, with 5,066 as S. aureus and 9,847 as CoNS. MALDI-TOF MS allowed the identification of approximately 85% of the CoNS strains, whereas only 14% of the CoNS strains were identified to the species level with phenotypic methods because they were often considered contaminants. Furthermore, the use of MALDI-TOF MS revealed the occurrence of recently characterized Staphylococcus species, such as S. pettenkoferi, S. condimenti, and S. piscifermentans. Microbiological relevance analysis further revealed that some species displayed a high rate of microbiological significance, i.e., 40% of the S. lugdunensis strains included in the analysis were associated with infection risk. This retrospective microbiological study confirms the role of MALDI-TOF MS in clinical settings for the identification of staphylococci with clinical consequences. The species distribution reveals the occurrence of the recently identified species S. pettenkoferi and putative virulent species, including S. lugdunensis. C oagulase-negative staphylococci (CoNS) are normal inhabitants of the human skin that lately have revealed themselves as significant etiological agents causing nosocomial infections, particularly in medical devices (1-4). Until recently, the phenotypic identification of Ͼ40 currently recognized CoNS species remained a challenge, because the technique was labor-intensive and uncommon CoNS were usually not identified (5, 6). Until recently, CoNS identification at the species level was performed only for clinical isolates that were considered clinically significant according to clinical or microbiological criteria. Meanwhile, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable, fast, and cost-effective alternative for bacterial species identification and is now widely used in microbiology laboratories (7,8). This method allows the identification of all staphylococci isolated in the clinical setting, thus rendering the use of well-known tests, such as free or bound coagulase tests, unnecessary. Previous reports have shown that MALDI-TOF MS is a reliable method to identify Staphylococcus at the species level; however, questions regarding the impact of such methods in clinical settings remain uncertain (9). Staphylococcus epidermidis is probably the major CoNS species causing nosocomial inf...