If environmental risk assessments for genetically modified lettuce (Lactuca sativa L.) are conducted in Japan, hybridization of native closely related species with lettuce needs to be evaluated, and 5 native species, L. indica, L. raddeana, L. sibirica, L. sororia, and L. triangulata, have been known to grow in Japan. In this study, we first screened EST-SSR markers with polymorphisms between the native species and lettuce using previously reported EST-SSR markers in order to develop DNA markers which can determine interspecific hybrid individuals. Next, hybridization of 3 species (L. sibirica, L. sororia, and L. triangulata) with lettuce was evaluated by pollination. Hybridization experiments were not conducted for the other species (L. indica and L. raddeana) because they have already been reported not to be crossed with lettuce. No hybrid individual was obtained by crossing lettuce with L. sororia or L. triangulata. On the other hand, hybrid individuals were obtained by crossing lettuce with L. sibirica; however, the hybrids stopped growing after germination. These results suggest that hybridization of the native species with lettuce would not occur in nature or that hybrids would not survive even if hybrids are produced in Japan.