The level of transgene expression in crop × weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T1 single-locus insert GFP/Bacillus thuringiensis (Bt) transgenic canola (Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC1F1, BC2F1) were produced by backcrossing various GFP/Bt transgenic canola (B. napus, cv Westar) and birdseed rape (Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC2F2 Bulk) were generated by crossing BC2F1 individuals in the presence of a pollinating insect (Musca domestica L.). The ploidy of plants in the BC2F2 Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F1 hybrid generations contained 95?97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15?29% presence in the BC2F2 Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC2F2 Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F1, BC1F1 and BC2F1). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.