Technical Abstract: Bacterial illness attributed to Campylobacter accounts for a high percentage of the estimated 77 million yearly incidents of food-borne illness in the USA. Of the 16 species of Campylobacter identified to date, 7 have been identified as potential human pathogens: C. jejuni, C. coli, C. lari, C. fetus, C. upsaliensis, C. sputorum and C. concisus. In consequence, there is a need for a simple, rapid, rugged, sensitive and specific technique for identifying Campylobacter species. MALDI-TOF-MS analyses of whole cell lysates provide a relatively simple method of species, sub-species and strain differentiation by identification of unique protein biomarkers. Several species of Campylobacter, namely C. jejuni, C. coli, C. lari, C. fetus, C. upsaliensis, C. sputorum and C. concisus were isolated and distinguishing biomarkers tentatively identified by MALDI-TOF-MS. One biomarker, a DNA-binding protein (HUP), of C. jejuni, C. coli, C. lari and C. upsaliensis was isolated and unambiguously identified by comparison of the LC/MS/MS analysis of its tryptic peptides to the protein sequence of the HUP gene from an internal Campylobacter species database generated by genomic sequencing. In addition, ESI/MS molecular weight of the isolated HUP was compared to the hypothetical molecular weight of HUP in the database. The HUP protein, and other protein biomarkers, provide excellent differentiation of Campylobacter species and, in some cases, Campylobacter strains. An unexpected advantage of definitive identification of the HUP biomarker ion was that the +1 and +2 charge states provided a two-point internal calibrant increasing the accuracy of molecular weight measurements of other biomarkers in the MALDI-TOF-MS spectra.