Gene flow is an important index for environmental biosafety assessments. Pollens are potential carriers for genetically modified DNA to other plants. For phanerogams, pollen viability and cross-compatibility are key factors for successful outcross hybridization. In this study, broccoli (Brassica oleracea var. italica) transformed with the ipt gene, designated as line 103 was used to investigate its potential outcross by pollen viability test and pollination with non-transgenic broccolis (line 104 and cv. ‘Green King’) and closely 5 related varieties, such as Chinese kales (B. oleracea var. alboglabra) ‘Fu Yue’ (white flower) and ‘Bai Ge Lin’ (yellow flower), cauliflower (B. oleracea var. botrytis) ‘Li Syue’, Pakchoi (B. rapa var. chinensis), and edible rape (B. rapa). These results provide valuable information for isolation and co-cultivation systems. Under field condition, flowering periods of transgenic ipt broccoli and non transgenic recipients were overlapping by 3–28 days in 2007 and 8–44 days in 2008. The pollen viability of F1 hybrids was determined by TTC (triphenyl tetrazolium chloride) method, FDA (fluorescein diacetate assay) and in vitro germination. The FDA showed no significant difference (p&amp;#65310;0.05) in pollen viability among transgenic line 103, non-transgenic and other tested cultivars. However, the differences of pollen viabilities were significant (p&lt;0.05) using TTC and in vitro germination. This result indicated that the cross abilities of transgenic and their hybridized offsprings were still existing. The range of hybridization rates were 86–100%, 1.1–42.5% and 0–9% by hand, insect (bee), and natural pollination, respectively. No hybridization was found from Pakchoi and edible rape as pollen recipients. The results showed that transgenic DNA insertion did not alter potential risk for outcross among broccoli varieties; however, there is possibility of gene transfer between transgenic and non-transgenic broccoli varieties.