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Koudelka Š., Gelbíčová T., Procházková M., Karpíšková R. (2018): Lineage and serotype identification of Listeria monocytogenes by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Czech J. Food Sci., 36: 452–458. The identification of Listeria species, lineages and serotypes remains a crucial issue not only in epidemic surveys, but also in monitoring of the diversity of bacteria in the food chain. The aim of this study was identification of L. monocytogenes strains at lineage and serotype level using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The performance of MALDI-TOF MS was tested to identify L. monocytogenes into two lineages (I and II) and four serotypes (1/2a, 1/2b, 1/2c and 4b) the most commonly found in humans and food. Total of 227 L. monocytogenes strains from different sources were subjected to the study. Some of strains (112) were used for main spectrum profile (MSP) library creation. Other strains of interest (115) were then correctly identified on the lineage level comparing with the library by MALDI-TOF MS analysis using Biotyper (90%) and ClinPro Tools (100%) software. The serotype identification with 55.7% (Biotyper) and 67.8% (ClinPro Tools) accuracy is rather a proof that under given conditions the method has not big potential to be used for serotyping. However, MALDI-TOF MS has a potential to identify lineages of L. monocytogenes of food and human origin.