When cultivating genetically modified varieties, the spontaneous gene flow between crop and wild relatives could be of concern. We analyzed spontaneous hybridization between a transgenic male-sterile line of oilseed rape (Brassica napus, 2n=38, AACC) and, as pollen donors, three European populations of wild radish (Raphanus raphanistrum, 2n=18, Rr,Rr) and a variety of cultivated radish (Raphanus sativus, 2n=18, RR). Seeds showed size and shape dimorphism that correlated to the frequency of hybrids. The offspring were scored morphologically and analyzed using DNA markers (inter-simple sequence repeats) to quantify hybrid frequencies. Seed set ranged from 0.4–1.2 seeds per pod, and 0.02–0.6 seeds per pod were confirmed as hybrids. The frequency of confirmed hybrids differed significantly among populations of R. raphanistrum. In the cross with a French population, all offspring were hybrids; in the cross with a Swiss population, 53% of the offspring were hybrids; and in the cross with a Danish population, only 2% of the offspring were found to be hybrids. The remaining offspring apparently belonged to two groups: the majority was B. napus-like plants, possibly of matromorphic origin, and a minority from the Danish cross seemed to carry fragments of the Raphanus genome. In the cross with a cultivated R. sativus, all offspring were found to be hybrids. This is the first report on spontaneous hybridization between B. napus and R. sativus. Hybrids from all cross-combinations had low pollen fertility (0–15%). If R. raphanistrum occurs where male-sterile B. napus is cultivated, large regional differences in hybridization frequencies between the species could complicate environmental risk assessment of transgenic oilseed rape.