Previous hybridisation studies showed that the repetitive DNA sequence pBNBH35 from Brassica nigra (genome BB, 2n=16) bound specifically to the B-genome and not to the A- or C-genomes of Brassica species. We amplified a sub-fragment of pBNBH35 from B. nigra by PCR, cloned and sequenced this sub-fragment, and confirmed that it was a 329-bp sub-fragment of pBNBH35. PCR and hybridisation techniques were used to confirm that the pBNBH35 sub-fragment was Brassica B-genome-specific. Fluorescence in situ hybridisation (FISH) in B. nigra, B. juncea (AABB, 2n=36) and B. napus (AACC, 2n=38) showed that the pBNBH35 sub-fragment was present on all eight Brassica B-genome chromosomes and absent from the A- and C-genome chromosomes. The pBNBH35 repeat was localised to the centromeric region of each B-genome chromosome. FISH clearly distinguished the B-genome chromosomes from the A-genome chromosomes in the amphidiploid species B. juncea. This is the first known report of a B-genome repetitive marker that is present on all B-genome chromosomes. It will be a useful tool for the detection of B chromosomes in interspecific hybrids and may prove useful for phylogenetic studies in Brassica species.