Although diphtheria has to be diagnosed primarily on clinical symptoms, the rapid and reliable detection and identification of the potentially toxigenic Corynebacterium species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis, is essential for the definite diagnosis and management of diphtheria with respect to both the individual patient and the public health measures to be undertaken. Laboratory confirmation of suspected diphtheria has to aim for the isolation of the etiologic pathogen (including species identification and antibiotic susceptibility testing) as well as for differentiation of toxigenic from non-toxigenic strains (by using tox gene detection and toxigenicity testing). The recent introduction of the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) into the microbiological algorithm of laboratory diagnosis of diphtheria allows the specific and escalating identification of the three potentially toxigenic Corynebacterium species from growing colonies. Finally, molecular typing techniques may be applied to explore the clonal relatedness of clinical isolates and their potential routes of transmission. The most relevant laboratory procedures fulfilling these requirements (microbiological culture, conventional biochemical tests, molecular methods for species identification and toxigenicity testing) will be presented here.