Isatis tinctoria var. yezoensis (Ohwi) Ohwi (Cruciferae) is one of major natural dyeing crops in the world and also have used as a medicinal plant in Korea. We evaluated seed purity in $F_1$-hybrid accessions using amplified fragment length polymorphism (AFLP) markers. One hundred sixty seeds from the male and female harvests were subsequently screened for seed purity with ten primers. The 13 accession-specific bands and many variable AFLP bands scored for accessions. Especially, E-AAC/M-CAA and E-AAG/M-CAT were presented clear hybrid bands for $F_1$ hybrids. $F_1$ hybrids maintained higher average level of genetic diversity compared with their correspondent parents. Self-inbred seeds from the female and male harvests were revealed 8.0% and 5.0%, respectively. The AFLP may lead to a better insight in to the hybrid seed purity test in I. tinctoria var. yezoensis.