In bread wheat, the genetic diversity of the D subgenome is less than that of the other subgenomes, because the chance hybridization events that created Triticum aestivum involved few Aegilops tauschii individuals. The diversity was further depleted through its subsequent domestication and breeding. Synthetic hexaploid wheat (SHW) lines are pre-breeding germplasm that can overcome this genetic bottleneck through accessing the wide genetic diversity of the Ae. Tauschii genepool. However, the phenotypes observed in the diploid Ae. Tauschii parents are not always recovered in the SHW lines, possibly due to subgenome interactions. In an attempt to understand the effect of post-polyploidization genome reprogramming at the transcriptome level, we performed RNA sequencing of four SHW lines and their tetraploid and diploid parents, across ten tissues and three biological replicates (240 samples). Homoeolog expression bias analysis using 18,357 triads suggested massive suppression of the D homoeoalleles in SHW lines when compared to their parental expression levels. Differential expression analysis of the whole-genome gene set also revealed extensive suppression of genes in the D subgenome. The qualitative differences of transcripts were unravelled through alternative splicing analysis, which indicated an additional layer of complexity where all five splice events were identified, and where ‘retained intron’ was predominant.