Technical Abstract: Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carboxyl-carrier protein. In this research, we cloned four genes encoding BC from Brassica napus (namely BnaC.BC.a, BnaC.BC.b, BnaA.BC.a and BnA.BC.b), and two from each of the two parental species, B. rapa (BraA.BC.a and BraA.BC.b) and B. oleracea (BolC.BC.a and BolC.BC.b). Sequence analyses revealed that in B. napus the gene BnaC.BC.a and BnaC.BC.b were from the C genome of B. oleracea, whereas BnaA.BC.a and BnaA.BC.b were from the A genome of B. rapa. Comparative and cluster analysis indicated that these genes were divided into two major groups, BnaC.BC.a, BnaA.BC.a, BraA.BC.a and BolC.BC.a in group-1, and BnaC.BC.b, BnaA.BC.b, BraA.BC.b, and BolC.BC.b in group-2. The divergence of group-1 and group-2 genes occurred in their common ancestor 13~17 million years ago (MYA), soon after the divergence of A. thaliana and Brassica (15-20 MYA) and identical to the previously reported triplicated time of paralogous subgenomes of diploid Brassica species and the divergence date of group-1 and group-2 genes of a-CT, another subunit of heteromeric ACCase, in Brassica. RT-PCR revealed that the expression level of group-1 and group-2 genes varied in different organs, and the expression patterns of the two groups of genes were similar in different organs, except in flower. The amino acid sequences of proteins encloded by these genes were highly conserved except the sequence encoding predicted plastid transit peptides. The plastid transit peptides on the BC precursors of Brassica (71-72 amino acid residues) were predicted based on AtBC protein, compared and confirmed by fusion with GFP. Our results will be helpful in elucidating the evolution and the regulation of ACCase in oilseed rape.