To understand the effects of habitat characteristics on the genetic diversity of Persicaria thunbergii, three sites of different environmental conditions in a water system were surveyed. Site A was the closest to the source of the water system, and there was a dam between sites A and B. Site C is located on the lowest downstream in the water system. Vegetation survey of four quadrats at each site was performed, and soil samples were collected for physicochemical analysis. Random amplification of polymorphic DNA (RAPD) analysis of ten P. thunbergii individuals at each site was conducted to calculate population genetic diversity and genetic distance among populations. Soil was sterile sand at site A, whereas loamy soil at sites B and C. A pure stand of P. thunbergii appeared at site A, while other species occurred together (such as Humulus japonicus and Phragmites australis) at sites B (ShannonWiener index; H B = 0.309) and C (H C = 0.299). Similar to the species diversity, genetic diversity (Nei's gene diversity; h) within population of site A (h A = 0.2381) was relatively lower than sites B (h B = 0.2761) and C (h C = 0.2618). However, site C was separated from sites A and B in genetic distance rather than the geographical distance (Nei's genetic distance; A~B, 0.0338; B~C, 0.0685; A~C, 0.0833).