Clostridium difficile is a pathogen on the move, as evidenced by the rapid transcontinental spread of the so-called hypervirulent 027 strains, followed by the emergence of further PCR ribotypes such as 017, 078 and 106. This provides a rare opportunity to study the evolution of virulence in action. However, to fully exploit this opportunity, robust phylogenetic methods on a diverse set of characterised strains are required to provide a reference evolutionary framework to study C. difficile epidemiology, ecology and virulence. Traditional phylogenetic classification of bacteria to study evolutionary relatedness is based on the characterisation of a limited number of genes, rRNA or signature sequences. However, due to the acquisition of DNA through lateral gene transfer, the differences between closely related bacterial strains can be vast. By contrast, whole genome sequencing comparisons allow all genes to be compared. Nevertheless, whole-scale genome sequencing remains an expensive endeavour and such comparisons are limited to only a handful of strains. DNA microarrays represent an alternative technology for whole genome comparisons enabling a “birds eye view” of all the genes absent or present in a given genome as compared to the reference genome on the microarray.