Oilseed rape ( Brassica napus L.) is an amphidiploid species that originated from a spontaneous hybridisation of Brassica rapa L. (syn. campestris) and Brassica oleracea L., and contains the complete diploid chromosome sets of both parental genomes. The metaphase chromosomes of the highly homoeologous A genome of B. rapa and the C genome of B. oleracea cannot be reliably distinguished in B. napus because of their morphological similarity. Fluorescence in situ hybridisation (FISH) with 5S and 25S ribosomal DNA probes to prometaphase chromosomes, in combination with DAPI staining, allows more dependable identification of Brassica chromosomes. By comparing rDNA hybridisation and DAPI staining patterns from B. rapa and B. oleracea prometaphase chromosomes with those from B. napus, we were able to identify the putative homologues of B. napus chromosomes in the diploid chromosome sets of B. rapa and B. oleracea, respectively. In some cases, differences were observed between the rDNA hybridisation patterns of chromosomes in the diploid species and their putative homologue in B. napus, indicating locus losses or alterations in rDNA copy number. The ability to reliably identify A and C genome chromosomes in B. napus is discussed with respect to evolutionary and breeding aspects.