Species of Brassica have small, morphologically similar chromosomes, which makes karyotyping difficult using conventional cytogenetic methods. Molecular cytogenetic methods, like fluorescence in situ hybridisation (FISH) have the potential to improve karyotyping through the use of chromosome- or genome-specific markers. Simultaneous application of more than one DNA probe can greatly enrich the results obtained compared with separate single target FISH experiments. This paper demonstrates the use of multicolour fluorescence in situ hybridisation with 5S and 25S rDNA for karyotyping three amphidiploid species: B. napus, B. juncea and B. carinata. Using this method, it was possible to identify eight out of nineteen pairs of chromosomes in B. napus, ten out of eighteen pairs in B. juncea and six out of sixteen pairs in B. carinata. Additionally, rDNA sites allow the determination of the genomic origin of all marked chromosomes in B. napus and B. juncea.