MALDI-TOF-MS of microorganisms, which identifies microbes based on masses of high abundant low molecular weight proteins, is rapidly advancing to become another standard method in clinical routine laboratory diagnostics. Allelic isoforms of these proteins result in varying masses of detectable biomarker ions. These variations give rise to a novel typing method for microorganisms named mass spectrometry-based phyloproteomics (MSPP). The base of MSPP is an amino acid sequence list of allelic isoforms caused by non-synonymous mutations in biomarker genes, which were detectable as mass shifts in an overlay of calibrated MALDI-TOF spectra. Thus, for each isolate a combination of amino acid sequences can be deduced from the scheme of recordable biomarker masses. Performing comparably to laborious multilocus and whole genome sequence typing (wgMLST)-approaches it is feasible to build phyloproteomic dendrograms using hierarchical cluster analysis. MSPP bears a high potential especially for identification of chromosomal localised virulence or antimicrobial resistance factors associated with evolutionary relatedness. In this study the principle of MSPP-typing was demonstrated on a Campylobacter jejuni ssp. jejuni isolate collection and MSPP was compared to MLST.