The multiplicity of cellular infection (MOI) is the number of virus genomes of a given virus species that infect individual cells. This parameter chiefly impacts the severity of within-host population bottlenecks as well as the intensity of genetic exchange, competition, and complementation among viral genotypes. Only a few formal estimations of the MOI currently are available, and most theoretical reports have considered this parameter as constant within the infected host. Nevertheless, the colonization of a multicellular host is a complex process during which the MOI may dramatically change in different organs and at different stages of the infection. We have used both qualitative and quantitative approaches to analyze the MOI during the colonization of turnip plants by Turnip mosaic virus. Remarkably, different MOIs were observed at two phases of the systemic infection of a leaf. The MOI was very low in primary infections from virus circulating within the vasculature, generally leading to primary foci founded by a single genome. Each lineage then moved from cell to cell at a very high MOI. Despite this elevated MOI during cell-to-cell progression, coinfection of cells by lineages originating in different primary foci is severely limited by the rapid onset of a mechanism inhibiting secondary infection. Thus, our results unveil an intriguing colonization pattern where individual viral genomes initiate distinct lineages within a leaf. Kin genomes then massively coinfect cells, but coinfection by two distinct lin-eages is strictly limited. IMPORTANCE The MOI is the size of the viral population colonizing cells and defines major phenomena in virus evolution, like the intensity of genetic exchange and the size of within-host population bottlenecks. However, few studies have quantified the MOI, and most consider this parameter as constant during infection. Our results reveal that the MOI can depend largely on the route of cell infection in a systemically infected leaf. The MOI is usually one genome per cell when cells are infected from virus particles moving long distances in the vasculature, whereas it is much higher during subsequent cell-to-cell movement in mesophyll. However, a fast-acting superinfection exclusion prevents cell coinfection by merging populations originating from different primary foci within a leaf. This complex colonization pattern results in a situation where within-cell interactions are occurring almost exclusively among kin and explains the common but uncharacterized phenomenon of genotype spatial segregation in infected plants.