We used polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques to demonstrate the presence of Brassica B-genome chromosomes and putative B-genome introgressions in B. napus x B. juncea interspecific progeny., The B-genome - specific repeat sequence pBNBH35 was used to generate PCR products and FISH probes. The highest frequencies of viable progeny were obtained when B. napus was the maternal parent of the interspecific hybrid and the first backcross. B-genome - positive PCR assays were found in 34/51 fertile F-2 progeny (67%), which was more than double the proportion found in fertile BC, progeny. Four B-genome - positive F-2-derived families and I BC1-derived family were fixed or segregating for B. juncea morphology in the F-4 and BC1S2, respectively, but in only 2 of these families did B. juncea-type plants exhibit B. juncea chromosome count (2n = 36) and typical B-genome FISH signals on 16 chromosomes. The remaining B. juncea-type plants had B. napus chromosome count (2n = 38) and no B-genome FISH signals, except for 1 exceptional F-4-derived line that exhibited isolated and weak B-genome FISH signals on 11 chromosomes and typical A-genome FISH signals. B. juncea morphology was associated with B-genome - positive PCR signals but not necessarily with 16 intact B-genome chromosomes as detected by FISH. B-genome chromosomes tend to be eliminated during selfing or backcrossing after crossing B. juncea with B. napus, and selection of lines containing B-genome chromatin during early generations would be promoted by use of this B-genome repetitive marker.