Rose is a major ornamental plant, and a lot of cultivars with attractive morphology, color and scent have been generated by classical breeding. Recent progress of genetic modification produces a novel cultivar with attractive features. In both cases, a major problem is the gene-flow from cultivated or genetically modified (GM) plants to wild species, causing reduction of natural population. To investigate whether gene-flow occurs in wild species, molecular analysis with DNA markers with higher efficient technique is useful. Here we investigated the gene-flow from cultivated roses (Rosa×hybrida) to wild rose species planted in close distance in the field. The overlapping flowering periods and visiting insects suggest that pollens were transported by insects between wild and cultivated roses. We examined the germination ratio of seeds from wild species, and extracted DNA and checked with KSN and APETALA2 (AP2) DNA markers to detect transposon insertions. Using two markers, we successfully detected the outcross between wild and cultivated roses. For higher efficiency, we established a bulking method, where DNA, leaves or embryos were pooled, enabling us to that check the outcross of many plants. Our results suggest that wild species and garden cultivars can cross in close distance, so that they should be planted in distance, and checked the outcross with multiple DNA markers.