Understanding the potential for the evolution of herbicide tolerance in weeds following gene flow from transgenic crops is important for their management. The inter-specific hybrids formed between four Brassica juncea populations with different glyphosate-susceptibility and transgenic oilseed rape was studied, emphasizing on the possible population difference. Compared with both parents, the fitness-related components and photosynthetic capacity of hybrids decreased dramatically, but significant differences were observed among different hybrids. All F1 hybrids exhibited highly enhanced but similar herbicide tolerance level regardless of the wild B. juncea parent, indicating that genetically engineered herbicide tolerance may over-dominate phenotype ones by wild-crop hybridization. The analysis of parental loci transmission revealed a higher transfer ratio of male-specific loci detected in F1 hybrids, suggesting that oilseed rape genetic markers can be transferred at relatively high frequencies to the next generation. Therefore, higher transfer ratio of oilseed rape-specific loci, coupled with variation of populations in fitness-related parameters in F1 hybrids, could complicate environmental risk assessment of transgenic oilseed rape, especially in current agroecosystems with increasing application of glyphosate.