Persistent aquatic environmental reservoirs for Vibrio cholerae O1 are present in Asia; however, their existence and importance in other parts of the world remains controversial. The single-source introduction of toxigenic V. cholerae O1 in Haiti, in the aftermath of the 2010 earthquake, provided a unique opportunity to assess the potential role played by aquatic reservoirs in subsequent seasonal outbreaks. Whole genome sequence Bayesian phylogeography showed robust evidence of V. cholerae O1 evolution in riverine sites, through the establishment of reservoirs, during lull periods of the Haitian epidemic. Novel lineages emerged in the environment from sequential population bottlenecks, characterized by mutations in genes potentially involved in adaptive response. The data highlight the importance of the aquatic environment in overall persistence and evolution of this global pathogen.