The economically valuable oil crop Brassica napus (AACC, 2n=38), which arose from interspecific hybridization between the diploid ancestors Brassica rapa (AA, 2n=20) and Brassica oleracea (CC, 2n=18), has a complex genome.  More than 10% of the assembled sequences, most of which belong to the C subgenome, have not been anchored to the corresponding chromosome.  Previously, a complete set of monosomic alien addition lines (MAALs, C1–C9) with each of the nine C-subgenome chromosomes added to the extracted A subgenome was obtained from the allotetraploid B. napus donor Oro, after the ancestral B. rapa (RBR Oro) genome was restored.  These MAALs effectively reduced the complexity of the B. napus genome.  Here, we determined the expression values of genes on unanchored scaffolds in the MAALs and RBR Oro.  Then, multiple comparisons of these gene expression values were used to determine the affiliations of the non-anchored scaffolds on which the genes were located.  In total, 54.68% (44.11 Mb) of the 80.67 Mb of non-anchored scaffolds belonging to the C subgenome were assigned to corresponding C chromosomes.  This work highlights the potential value of these MAALs in improving the genome quality of B. napus.