Aegilops umbellulata Zhuk. is a diploid wild wheat relative with UU genome. Ae. umbellulata has been utilized for bread wheat breeding through interspecific crossing with tetraploid wheat (Bansal et al. 2017). The grass-clump dwarfism (GCD), one of the hybrid incompatibilities between tetraploid wheat and Ae. umbellulata, is observed in 25% of triploid ABU hybrids triggered by epistatic interaction between AB and U genomes (Okada et al. 2017). Therefore, GCD acts as a reproductive barrier between tetraploid wheat and Ae. umbellulata, which inhibits production of synthetic allohexaploids with AABBUU genome. To identify the causal U-genome gene for GCD, RNA-seq-based bulked segregant analysis was performed. For generating mapping population, Triticum turgidum L. ssp. durum cv. Langdon (Ldn) and two accessions of Ae. umbellulata were used. An Ae. umbellulata accession showing GCD in the ABU hybrids was crossed with another one showing the normal hybrid phenotype. Then, pollens of the F1 plant of Ae. umbellulata were crossed to Ldn. We obtained 100 individuals of the ABU hybrids. RNA samples were extracted from young leaves of the parental Ae. umbellulata accessions and ABU hybrids, and two sets of bulks were mixed using RNAs from each hybrids showing GCD or the normal phenotype. SNP index was calculated based on the RNA-seq reads of each bulk. A large number of positions with high SNP index were densely distributed only to the distal region on the long arm of chromosome 6U. The CAPS markers were designed using the polymorphism information with high SNP index, and a linkage map of the 6UL chromosomal region was constructed. Then, the causal gene for GCD was successfully mapped on 6UL in the ABU hybrid population. On the other hand, microarray analysis of GCD showed that some APETALA1-like MADS-box genes, which could act as flowering promoters, were down-regulated, and that expression patterns of the miR156/SPLs module, which controls tiller number and blanching, were altered in crown tissues of the GCD exhibiting hybrids. Next, RNA sequencing analysis was conducted in crown tissues of the ABU hybrids. The expression patterns were compared in the GCD showing hybrids between the microarray and RNA-seq analyses. The RNA-seq reads were mapped to the causal D-genome region on 6UL, and the mapped reads were used to enrich the candidates. Our discovery would contribute to elucidating molecular mechanisms of postzygotic reproductive barriers and to utilize efficiently the Ae. umbellulata genes in further wheat breeding.