Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. Severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the strain NAP1; however, the existence of other severe disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicates reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that associate with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, allowed the determination that C. difficile has a core genome of 3.4 Mb, comprising ~3000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated on a multi-strain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel has associated patient data which allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 SNPs associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the gene sspA, was found to be identical across 177 isolates sequenced and lacked significant similarity to other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate better with disease severity than currently used diagnostic markers, as they were also present in the A-B+ strain type. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species-specific or associated with multiple severe disease strains.