Areas infested with Solanum carolinense, an introduced perennial weed, have increased quickly in Japan. The genetic structure of a S. carolinense population along the Takano River in Kyoto, Japan was investigated using amplified fragment length polymorphism (AFLP) analysis to reveal how introductions and local spread have contributed to the development of this population. Along a terrace beside the river and an adjacent roadside, we defined a cluster of above-ground shoots in the population as a subpopulation and 60 subpopulations were identified. Seventeen of these were selected and 165 shoots were analyzed by AFLP analysis using three primer pairs. The AFLP profiles revealed 69 genotypes and the presence of several clones, i.e. genotypes that were present in more than one individual. In total, 19 clones, comprising 2–34 identical genotypes, could be identified in the population. Each of these clones, except one, was allocated within each of the subpopulations. Clustering of the subpopulations was supported by high bootstrap values in all cases. Therefore, introductions from distant regions have mainly contributed to the development of this population, and local spread by seed or vegetative reproduction has rarely been important. The most likely introduction route of S. carolinense to this population was via planting associated with construction works.