Persistent herbicide application to weed species across vast areas promotes the rapid evolution of herbicide resistance among them. Ryegrass (Lolium spp) is one of the most reported herbicide-resistant weeds. Because ryegrass species is major weed of wheat field of wheat-exporting countries, their seeds are introduced into Japan as contaminants in imported wheat commodity. Herbicide resistant plants were detected in surrounding area of major ports. To assess diffusion risk of herbicide resistant gene around port area, we analyzed abundance of ALS inhibitor resistant individuals by PCR-RFLP and spatial genetic structure of the species by SSR (microsatellite) polymorphism. We also analysed frequency of resistant progenies directly in an intensive observation site by the produced seed. The frequency of resistant individuals was not changed for three years in every observed site. The percentage of resistant progeny was same as parents. These results suggest that the herbicide resistant gene is sustained through outcrossing within population, not only seeds spilling out from contaminants. Bayesian clustering analysis based on SSR detected two clusters. One cluster included populations close to the port where the resistant plants were detected. The contaminant seeds were assigned to the cluster. Another cluster included populations distributed more than 3 km from the port. Japanese ryegrass cultivars were assigned to this cluster. These results suggest that the port populations were derived from contaminant seeds, while the peripheral populations were originated from escaped cultivars.