During the past decade, sequencing of the entire genome of pathogenic bacteria has become a widely used practice in microbiology research. More recently, sequence data from multiple isolates of a single pathogen have provided new insights into the microevolution of a species as well as helping researchers to decipher its virulence mechanisms. The comparison of multiple strains of a single species has resulted in the definition of the species pan-genome, as a measure of the total gene repertoire that can pertain to a given microorganism. This concept can be exploited not only to study the diversity of a species, but also, as we discuss here, to provide the opportunity to use a knowledge-based approach for the development of novel vaccine candidates and new-generation targets for antimicrobials.