Cross-pollination and high cross-compatibility are frequently found in many cruciferous plants, which implies that there might be potential risk of alien gene flow among crucifers. In this work, the alien glyphosate-resistant gene flow between GM (Genetically modified) rapeseed variety Q3 (Brassica napus) and 6 varieties from 5 cruciferous species was assessed by sexual compatibility through aniline blue fluorescence observation, manual cross, mentor pollination with the mixture pollen and DNA identification. The results indicated that the compatibility indices of two B. napus varieties, B. juncea var. multiceps Tsen et Lee and Chinese cabbage (B. chinensis) (as female) with Q3 (as male) were 16.15, 12.77, 2.345 and 0.85, respectively. F1 plants were examined by PCR and all of these samples were confirmed as positive. Crossing between Orychophragmus violaceus, Descurainia sophia (as female) and Q3 (as male) expressed high cross-incompatibility. Fluorescence microscope observation indicated that growing of pollen tube terminated at the upper 1/3 part of the style of O. violaceus and on the stigma surface of D. sophia. The mentor plants were further analyzed by PCR and all were shown to be negative. Under the neighboring growth and natural pollination conditions, the rates of gene flow to two B. napus varieties were 2.3278% and 2.1487%, respectively, B. juncea var. multiceps Tsen et Lee and Chinese cabbage were 1.0157% and 0.9236%, respectively. No gene flow was observed in O. violaceus and D. sophia. Frequency of gene flow was highly correlated with sexual compatibility. Due to possible alien gene flow among crucifers, special consideration and care should be taken to grow GM rapeseed.