The significance of large tropical lakes as environmental reservoirs of Vibrio cholerae in cholera endemic countries has yet to be established. By combining large scale plankton sampling, microbial culture and ultrasensitive molecular methods, namely Droplet Digital PCR (ddPCR) and targeted genomics, the presence of Vibrio cholerae was investigated in a 96,600 L volume of surface water collected on a 322 nautical mile (596 km) transect in Lake Tanganyika. V. cholerae was detected and identified in a large area of the lake. In contrast, toxigenic strains of V. cholerae O1 or O139 were not detected in plankton samples possibly in relation to environmental conditions of the lake ecosystem, namely very low salinity compared to marine brackish and coastal environments. This represents to our knowledge, the largest environmental study to determine the role of tropical lakes as a reservoir of V. cholerae.