Abstract Background Clostridium difficile is a Gram-positive, anaerobic, spore-forming bacterium that is responsible for C. difficile associated disease in humans and is currently the most common cause of nosocomial diarrhoea in the western world. This current status has been linked to the emergence of a highly virulent PCR-ribotype 027 strain. The aim of this work was to identify regions of sequence divergence that may be used as genetic markers of hypervirulent PCR-ribotype 027 strains and markers of the sequenced strain, CD630 by array comparative hybridisation. Results In this study, we examined 94 clinical strains of the most common PCR-ribotypes isolated in mainland Europe and the UK by array comparative genomic hybridisation. Our array was comprehensive with 40,097 oligonucleotides covering the C. difficile 630 genome and revealed a core genome for all the strains of 32%. The array also covered genes unique to two PCR-ribotype 027 strains, relative to C. difficile 630 which were represented by 681 probes. All of these genes were also found in the commonly occuring PCR-ribotypes 001 and 106, and the emerging hypervirulent PCR-ribotype 078 strains, indicating that these are markers for all highly virulent strains. Conclusions We have fulfilled the aims of this study by identifying markers for CD630 and markers associated with hypervirulence, albeit genes that are not just indicative of PCR-ribotype 027 strains. We have also extended this study and have defined a more stringent core gene set compared to those previously published due to the comprehensive array coverage. Further to this we have defined a list of genes absent from non-toxinogenic strains and defined the nature of the specific toxin deletion in the strain CD37.