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Currently, Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) is being evaluated for its efficacy as a fast bacterial typing tool due to its great speed compared to other molecular methods. In this study, we evaluated MALDI-TOF as a tool for quick identification and typing of Francisella tularensis. This study encompassed 86 strains from two different geographical origins (Spain and the Czech Republic), which were previously characterised by Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA). The direct colony method was used for microbial identification. High-quality spectra of the 86 strains were obtained and their main spectra profiles (MSPs) were created for epidemiological typing using MALDI-TOF. Based on the MSPs, principal components were generated and a dendrogram was constructed. An in-house MALDI-TOF library entry was created for each group of PFGE and MLVA strains based on their high-quality spectra. Two dendrograms were obtained using these entries and the unique peaks in each entry were searched. All strains were correctly identified to the species level. No clear divisions were found in the 86-strain dendrogram; however, Spanish and Czech strains appeared separately in dendrograms created using MLVA and PFGE entries. Entries from our in-house MALDI-TOF library revealed 2–4 biomarker peaks for the detection of the five PFGE groups and 1–12 biomarker peaks for the detection of the seven MLVA groups. Finally, two and one specific biomarkers were found in the Czech and Spanish strains, respectively. MALDI-TOF can be used to accurately identify F. tularensis strains in less than 15 min. Moreover, data on geographical origin and PFGE and MLVA groups could be obtained in less than one hour after colony growing. • Mass Spectrometry correctly identifies Francisella tularensis strains in less than 15 min, even if protein extraction is not performed. • A reliable clustering could not be achieved by the dendrogram obtained of all strains. • Dendrograms generated from the entries of our in-house library could be useful for the determination of the geographical origin of F. tularensis strains. [ABSTRACT FROM AUTHOR]