Interspecific hybridization between two different Brassicaceae species, namely Brassica rapa ssp. pekinensis (♀) (AA, 2n = 2x = 20) and genetically modified Brassica napus (♂) (AACC, 2n = 4x = 38), was performed to study the transmission of a herbicide resistance gene from a tetraploid to a diploid Brassica species. Initially, four different GM B. napus lines were used for hybridization with B. rapa via hand pollination. Among the F1 hybrids, the cross involving the B. rapa (♀) × GM B. napus (♂) TG#39 line exhibited the highest recorded crossability index of 14.7 ± 5.7. However, subsequent backcross progenies (BC1, BC2, and BC3) displayed notably lower crossability indices. The F1 plants displayed morphological characteristics more aligned with the male parent B. napus, with significant segregation observed in the BC1 generation upon backcrossing with the recurrent parent B. rapa. By the BC2 and BC3 generations, the progeny stabilized, manifesting traits from both parents to varying degrees. Cytogenetic analysis revealed a substantial reduction in chromosome numbers, particularly in backcrossing progenies. BC1 plants typically exhibited 21-25 chromosomes, while BC2 progenies showed 21-22 chromosomes, and by the BC3 generation, stability was achieved with an average of 20 chromosomes. SSR marker analysis confirmed the progressive reduction of C-genome regions, retaining minimal C-genome-specific bands throughout successive backcrossing. Despite the extensive elimination of C-genome-specific genomic regions, the glyphosate resistance gene from the male parent B. napus was introgressed into BC3 progenies, suggesting that the glyphosate resistance gene located and introgressed in A-chromosome/genome regions of the Brassica plants.