is the causative agent of listeriosis, a severe foodborne illness characterized by septicemia, meningitis, encephalitis, abortions, and occasional death in infants and immunocompromised individuals. is composed of four genetic lineages (I, II, III, and IV) and fourteen serotypes. The aim of the current study was to identify proteins that can serve as biomarkers for detection of genetic lineage III strains based on simple antibody-based methods. Liquid chromatography (LC) with electrospray ionization tandem mass spectrometry (ESI MS/MS) followed by bioinformatics and computational analysis were performed on three strains (NRRL B-33007, NRRL B-33014, and NRRL B-33077), which were used as reference strains for lineages I, II, and III, respectively. Results from ESI MS/MS revealed 42 unique proteins present in NRRL B-33077 and absent in NRRL B-33007 and NRRL B-33014 strains. BLAST analysis of the 42 proteins against a broader panel of >80 sequenced strains from lineages I and II revealed four proteins [TM2 domain-containing protein (NRRL B-33077_2770), DUF3916 domain-containing protein (NRRL B-33077_1897), DNA adenine methylase (NRRL B-33077_1926), and protein RhsA (NRRL B-33077_1129)] that have no homology with any sequenced strains in lineages I and II. The four genes that encode these proteins were expressed in strain DE3 and purified. Polyclonal antibodies were prepared against purified recombinant proteins. ELISA using the polyclonal antibodies against 12 lineage I, II, and III isolates indicated that TM2 protein and DNA adenine methylase (Dam) detected all lineage III strains with no reaction to lineage I and II strains. In conclusion, two proteins including TM2 protein and Dam are potentially useful biomarkers for detection and differentiation of lineage III strains in clinical, environmental, and food processing facilities. Furthermore, these results validate the approach of using a combination of proteomics and bioinformatics to identify useful protein biomarkers.