The study of prokaryotic chromosome segregation has focused primarily on bacteria with single circular chromosomes. Little is known about segregation in bacteria with multipartite genomes. The human diarrhoeal pathogen Vibrio cholerae has two circular chromosomes of unequal sizes. Using static and time-lapse fluorescence microscopy, we visualized the localization and segregation of the origins of replication of the V. cholerae chromosomes. In all stages of the cell cycle, the two origins localized to distinct subcellular locations. In newborn cells, the origin of chromosome I (oriCIvc) was located near the cell pole while the origin of chromosome II (oriCIIvc) was at the cell centre. Segregation of oriCIvc occurred asymmetrically from a polar position, with one duplicated origin traversing the length of the cell towards the opposite pole and the other remaining relatively fixed. In contrast, oriCIIvc segregated later in the cell cycle than oriCIvc and the two duplicated oriCIIvc regions repositioned to the new cell centres. DAPI staining of the nucleoid demonstrated that both origin regions were localized to the edge of the visible nucleoid and that oriCIvc foci were often associated with specific nucleoid substructures. The differences in localization and timing of segregation of oriCIvc and oriCIIvc suggest that distinct mechanisms govern the segregation of the two V. cholerae chromosomes.