Key message <p>A complete set of monosomic alien addition lines (A^<i>n</i>A^<i>n</i>C^<i>n</i>C^<i>n</i> + B^<i>j</i>_1–8, 2<i>n</i> = 39) of B subgenome derived from <i>Brassica juncea</i> in <i>B. napus</i> enhancing the genetic stock of <i>B. napus</i> was developed and comprehensively analyzed.</p> Abstract <p>Monosomic alien addition lines (MAALs), in which an alien chromosome from a related species is introduced into the genome of the recipient plant, serve as valuable resources for crop genetic analysis and breeding. In this study, we developed a complete set of MAALs for the B subgenome of <i>Brassica juncea</i> (A^<i>j</i>A^<i>j</i>B^<i>j</i>B^<i>j</i>, 2<i>n</i> = 36) in the background of <i>B. napus</i> (A^<i>n</i>A^<i>n</i>C^<i>n</i>C^<i>n</i>, 2<i>n</i> = 38) through continuous backcrossing from BC₁ to BC₄ using <i>B. napus</i> and an octoploid progenitor (AABBCCRR, 2<i>n</i> = 72). Eight distinct MAALs (A^<i>n</i>A^<i>n</i>C^<i>n</i>C^<i>n</i> + B^<i>j</i>_1–8, 2<i>n</i> = 39), each carrying a different B subgenome chromosome added to the <i>B. napus</i> genome, were identified by PCR amplification of B chromosome-specific primers and fluorescence in situ hybridization (FISH). Morphologically, most MAALs show distinct differences from one another and exhibit varying degrees of phenotypic divergence compared to the parental <i>B. napus</i>, such as purple seedling leaves in AACC-B5 and a higher seed number per silique in AACC-B7. Moreover, extensive homoeologous exchange (HE) was detected in MAALs. Bivalent pairing involving the alien chromosome was observed in 20% (AACC-B6) to 42% (AACC-B2) of pollen mother cells (PMCs), and the pairing rates between the alien B chromosome and C genome chromosomes were significantly higher than those between the alien B and A genome chromosomes. Additionally, B chromosomes exhibited varying effects on A-C chromosome pairing in different addition lines, with pairing frequencies ranging from 0.73 (AACC-B8) to 1.68 (AACC-B1) for A-C chromosome associations. Transmission rates of the added B chromosomes via ovules were significantly higher than those via pollen, indicating preferential transmission through female gametes. This MAAL collection provides valuable genetic stock for analyzing B subgenome structure, establishes a foundation for studying gene interactions among the A, B, and C genomes, and offers novel germplasm resources for <i>B. napus</i> practical breeding.</p>