Research into the molecular mechanisms used by Corynebacterium diphtheriae to cause disease has been aided by a wide variety of innovative, recently developed, molecular, biochemical and genetic tools. Multiple compatible plasmid origins are now available and the methods used to introduce DNA to C. diphtheriae have been both improved and expanded to include conjugation. In the last decade, a technique to perform transposon mutagenesis in C. diphtheriae was developed as well as phage-based vectors that permit introduction of DNA at a known specific chromosomal site. Genetic systems that model C. diphtheriae gene regulation in E. coli have been invaluable tools and in vitro systems to characterize protein binding and to identify genes controlled by the diphtheria toxin repressor are available. Finally the recent expansion of sequencing and bioinformatics has yielded multiple genomic sequences for comparison both to each other and to the genomes of pathogens from other species. The vast expansion of tools available to characterize the pathogenic mechanisms of C. diphtheriae has been a boon to researchers; resulting in the identification of new virulence factors, including pili and a heme oxygenase, in a species that has been studied in laboratories for more than 100 years.