Technical Abstract: The human bacterial pathogen Campylobacter jejuni contains two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Although Cjd strains are isolated infrequently in many parts of the world, they are obtained primarily from human clinical samples and result in an unusual clinical symptomatology in that, in addtion to gastroenteritis, they are associated often with bacteremia. In this study, we describe a novel multiplex PCR method that can be used to unambiguously subspeciate C. jejuni isolates. This method correctly subspeciated 188 Cjj and Cjd isolates. In addition, combination of this multiplex PCR with a recently described lpxA-based speciation PCR method permits speciation and subspeciation of thermophilic Campylobacter spp. in a single amplification. We also present, for the first time, a genomic comparison of the two C. jejuni subspecies, using DNA microarray-based comparative genomic hybridization and MALDI-TOF mass spectrometry. Multiple metabolic, transport and virulence functions (e.g. cytolethal distending toxin) are shown to be variable between the two subspecies. Comparison of the two subspecies yields not only intriguing insights into potential differences in virulence but also provides clues as to the potential evolutionary origin of Cjd.