An improved modification of genomic in situ hybridization (GISH) was proposed. It allows clear and reproducible discrimination between closely related genomes of both tetraploid and hexaploid wheat species due to preannealing of labeled DNA probes and prehybridization of chromosomal samples with blocking DNA. The method was applied to analyze intergenomic translocations 6A:6B and 1A:6B identified in the IG46147 and IG116188 samples of tetraploid wheat Triticum dicoccoides by C-banding. The structure of the rearranged chromosomes was defined for two translocation variants, and the breakpoints were identified on the chromosome arms. Possible application of the developed GISH variant to study genome reorganizations during speciation of allopolyploid plants in evolution is discussed.