Phylogenomic structuring of the Vibrio cholerae clade is an important area of study in the context of the alarming spike of human morbidities due to environmental pathogens around the world. Even after decades of research, the structuring of natural populations of V. cholerae and its sister species is poorly understood. Ecological and epidemiological importance of the clade shouts for a sound understanding of the phylogenetic relationships and environmental dynamics within the clade in their natural settings. In this thesis, my goal is to answer few broad questions on the biology of the V. cholerae clade: i) How different is the evolutionary trajectory of pathogenic lineages from environmental populations ii) To what extent environmental and biogeographic factors impact the intra-species diversity of the model species: V. cholerae iii) Is our understanding of the phylogeny and ecology of the V. cholerae clade consistent with the inclusion of the recently discovered sister species of V. cholerae from both environmental and clinical settings. To address these questions, I have employed culture-based techniques, phenotypic characterization, high throughput amplicon sequencing, whole genome sequencing, quantitative PCR (qPCR) as well as phylogenetic and taxonomic inference tools. I have found potential signatures in V. cholerae population structure in cholera endemic and non-endemic regions. I have also shown the impact of environmental factors on the intra-species diversity of V. cholerae population naturally occurring in a cholera endemic area. Furthermore, I am describing here the genetic, phylogenetic and biological traits, as well as interaction of a novel sister species with V. cholerae: V. paracholerae sp. nov. Moreover, I delineate the phylogenetic and molecular spectrum of a novel sister species of V. cholerae: V. tarriae, isolated from human and environmental samples only from cholera free locations in the USA. The studies presented herein consolidates the understanding of the natural diversity and ecology of the clade containing V. cholerae and its close relatives at species and sub-species level, which could be a significant stride in our knowledge on this group of microorganisms.