In the present study a well-characterized strain collection (n = 33) of Avibacterium species was investigated by matrix-assisted laser desorption ionization-time-of flight mass spectrometry (MALDI-TOF MS). The robustness of the currently available reference database (Bruker Biotyper 3.0) was tested to determine the degree of identification of these strains. Reproducible signal patterns were obtained from all strains. However, identification of most strains was only possible at genus level. Furthermore, two strains could not be identified by this method. Based on their protein spectra profiles, a MALDI main spectra dendrogram was created to determine their relationship. Most strains were closely related-for example, 26 strains formed cluster 1 including the type strains of Avibacterium volantium, Avibacterium gallinarum, Avibacterium endocarditidis and Avibacterium avium-while Avibacterium paragallinarum biovars 1 and 2 formed cluster 2 and, finally, strain 55000 remained on its own. The present MALDI-TOF MS results confirm recent findings that only certain isolates of Av. paragallinarum represent a well-defined species within the genus Avibacterium, making a taxonomic revision essential. To improve identification of Avibacterium at species level by MALDI-TOF MS, relevant reference strains were included in the newly created database and results are presented. In conclusion, Av. paragallinarum can be identified by MALDI/Biotyper and not the other species of the genus.