Simultaneous genomic in situ hybridization with probe preannealing (SP-GISH) was used for discriminating Aegilops speltoides chromosome regions by their relatedness to DNA of other species. We used a hybridization mixture of two differently labelled DNAs, one from the species used for chromosome spread preparations and a second from species of different and varying affinity, thus creating a two-colour system showing chromosome regions where alien DNA hybridized. Genomic DNA from A. speltoides was labelled with biotin and preannealed with digoxigenin-labelled total genomic DNA from different accessions of Ae. speltoides, Ae. bicornis, Ae. tauschii and Hordeum spontaneum. The probe mixture was hybridized to mitotic chromosmes of Ae. speltoides. Chromosome regions of preferential hybridization of self-DNA were visualized as green, whereas regions of combined hybridization showed orange–yellow fluorescence. We observed GISH banding patterns with a different degree of green fluorescence along Ae. speltoides chromosomes that directly correlated with evolutionary distance. Small green bands were observed in subtelomeric and telomeric heterochromatic regions using DNA of a different accession of Ae. speltoides, whereas when using DNA of H.spontaneum most regions of the chromosomes, except pericentromeric regions, showed mainly green fluorescence. The resolution and application of the approach to the study of heterochromatin differentiation are discussed.