The waterborne pathogen Vibrio cholerae continues to be responsible for devastating global pandemics of cholera disease. Strains of the O1 or O139 serogroups that carry the cholera toxin can colonize the small intestine of humans and cause massive efflux of water from the victim, resulting in dehydration and death if treatment with saline fluids is not initiated quickly. However, the majority of strains isolated from environmental settings do not carry cholera toxin and are serogroups other than O1 or O139 (hence, their non-O1, non-O139 strain designation). Although these strains are often lumped together when discussing causative agents of cholera disease, they are in fact quite genotypically and phenotypically diverse. In Chapter 2 of this thesis, a first look at the types of genes that account for the genotypic diversity is presented, and this work demonstrated that the V. cholerae pan-species genome is quite extensive. Chapter 3 then follows up on the discovery of a gene encoding a toxin similar to exotoxin A of Pseudomonas aeruginosa, a potent secreted virulence factor of this opportunistic pathogen. In a joint publication, our work describing the biological effects of the new toxin, now designated cholix toxin, complements the work of our collaborators at the University of Guelph, who solved the crystal structure of both full length cholix toxin and its catalytic domain. Chapter 4 then takes a phylogenetic approach to understanding the role the toxin may be playing in V. cholerae virulence or environmental survival, by assessing the presence or absence of the gene encoding cholix toxin, chxA, in a collection of V. cholerae strains amassed from around the world. The chxA gene was then sequenced from a wide variety of clinical and environmental strains, revealing the presence of specific regions with extensive sequence variation. A final chapter describes further efforts to uncover the purpose of this toxin, as well as ideas for future research directions to pursue this goal. Cholix toxin provides a model for understanding selective pressures and the evolution of toxin genes in environmental settings.