Restriction landmark genome scanning (RLGS) is a high-speed genome analysis method to detect DNA polymorphism, and is suitable to develop useful DNA markers for cultivar-discrimination of agricultural plants. The mat-rush, Juncus effusus is a vegetatively propagated crop that has been cultivated for over 500 years in Japan as the material of “tatami-omote,” a surface of Japanese conventional mats. In recent years, Japanese mat-rush cultivars are grown in foreign countries and reimported to Japan in violation of the Seeds and Seedlings Law, economically damaging domestic production. The largest problem of illegal mat-rush reimportation has been with cv. Hinomidori. We developed a cultivar-discrimination DNA marker of mat-rush using RLGS, and detected the RLGS spot markers that didn′t exist specifically in Hinomidori. The spot marker can be used with the RLGS method to identify Hinomidori accurately among cultivars.