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RNA interference (RNAi) refers to a common mechanism of RNA-based post-transcriptional gene silencing in eukaryotic cells. In model plant species such as Arabidopsis and rice, RNAi has been routinely used to characterize gene function and to engineer novel phenotypes. In polyploid species, this approach is in its early stages, but has great potential since multiple homoeologous copies can be simultaneously silenced with a single RNAi construct. In this article, we discuss the utilization of RNAi in wheat functional gene analysis and its effect on transcript regulation of homoeologous genes. We also review recent examples of RNAi modification of important agronomic and quality traits in wheat and discuss future directions for this technology.