This study has employed multicolour fluorescence in situ hybridisation (MCFISH) and Diversity Arrays Technology (DArT) markers to determine the segregation of parental A, B and D genome material into the progeny of a cross between a hexaploid bread wheat (Triticum aestivum L. var. 2-49) and a tetraploid durum wheat [T. turgidum L. spp. durum (Desf.) var. Bellaroi]. In the F₂ progeny from a 2-49/Bellaroi cross, 82 out of 83 F₂ plants investigated with DArT analysis carried some D genome material, principally as entire chromosomes, while 40 plants included at least one complete copy of all seven D genome chromosomes. Twelve plants containing partial D chromosomes were identified. MCFISH analysis of 26 additional F₂ plants of the same cross showed that all 26 plants contained varying amounts of D genome material of which three carried single A-D translocations. In addition two telocentric D genome chromosomes were detected. The D genome content of each line and the breakpoint positions of the three A-D translocations were confirmed with DArT marker analysis. Overall results indicate a random recombination of A and B genome loci from the hexaploid female parent and the tetraploid male parent in this F₂ population and a significant retention of the maternal D genome material. This study illustrates that the combined application of the MCFISH and DArT techniques provides a powerful approach for the analysis of crosses between cereal genotypes of different ploidy.