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Misidentification between Taylorella equigenitalis , the causative agent of contagious equine metritis (CEM), and Taylorella asinigenitalis is observed by the gold standard culture method. The performance of matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) for Taylorella species identification was evaluated using 85 T. equigenitalis and 28 T. asinigenitalis strains selected on the basis of multilocus sequence typing data. Seven of the T. equigenitalis and 9 of the T. asinigenitalis strains were used to generate in-house reference spectra to expand the existing commercial Bruker database. Two bacterial incubation times and 3 different sample preparation procedures were compared. Overall, we demonstrated the usefulness of MALDI-TOF MS as a differential diagnostic tool for CEM; however, commercial spectra databases should be expanded with T. asinigenitalis reference spectra to achieve the expected performance. Moreover, direct spotting of 48-h colonies was not only the most efficient protocol but also the easiest to implement in a clinical setting. • An assessment of the reliability of MALDI-TOF MS for the identification of Taylorella species is presented. • Different bacterial incubation times and sample preparation procedures are compared. • Species-level identification of Taylorella equigenitalis is demonstrated. • Species-level identification of Taylorella asinigenitalis is demonstrated when the Bruker database is expanded with in-house T. asinigenitalis reference spectra. [ABSTRACT FROM AUTHOR]