Specific markers for Coxiella burnetii (C.b.) isolates RSA 493, Priscilla, and BUD were detected using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The method revealed noticeable differences in the ion signal profiles of the isolates in the mass range of 318 kDa. The number of characteristic ions for RSA 493, BUD, and Priscilla was 24, 15, and 7, respectively. The specific markers were compared against C.b. database using the Tag-Ident proteomics tool. For the isolates RSA 493, Priscilla and BUD there were identified 11, 5 and 3 potential biomarkers, respectively. This method represents a powerful tool for the rapid, sensitive, and differential characterization of C.b. isolates and is a good candidate for phyloproteomic approaches.