The Brassica napus-B genome monosomic addition lines (MALs) (AACC + B`, 2n = 39) were developed from self-pollination of pentaploid hybrids (AABCC) that were derived from hybridization between hexaploid hybrids (AABBCC) and B. napus (AACC). The alien chromosomes of the B genome in MALs were identified by the GISH technique, by observation of the meiotic behavior of pollen mother cells (PMCs), and by B-genome-specific molecular marker analysis. Studies of the meiotic behavior of B. napus-B genome chromosome MALs at diakinesis revealed that the majority of the chromosome configuration was 19II+1I, which indicated that the alien B genome chromosome remained univalent in most cases. The laggard-free PMCs also appeared at a lower ratio, which indicated that the B genome chromosome could be transmitted into gametes. The chromosome configurations of 20II and 19II+2I that appeared in double MALs (AACC+ 2 chromosomes of the B genome) indicated different homoeology between different B genome chromosomes. The paired B genome bivalent in double MALs can be normally segregated at anaphase in most cases. PMCs with multivalents were observed in all the double MAL combinations, which indicated homology of the B genome chromosomes with the A or C genome chromosomes.