Advances in the genetic transformation of cereals have improved the prospects of using biotechnology for plant improvement, and a toolbox of promoters with defined specificities would be a valuable resource in controlling the expression of transgenes in desired tissues for both plant improvement and molecular farming. A number of promoters have been isolated from the important cereals (wheat, barley, rice and maize), and these promoters have been tested mostly in homologous cereal systems and, to a lesser extent, in heterologous cereal systems. The use of these promoters across the important cereals would add value to the utility of each promoter. In addition, promoters with less sequence homology, but with similar specificities, will be crucial in avoiding homology-based gene silencing when expressing more than one transgene in the same tissue. We have tested wheat and barley promoters in transgenic barley and wheat to determine whether their specificity is shared across these two species. The barley bifunctional α-amylase/subtilisin inhibitor (Isa) promoter, specific to the pericarp in barley, failed to show any activity in wheat, whereas the wheat early-maturing (Em) promoter showed similar activity in wheat and barley. The wheat high-molecular-weight glutenin (HMW-Glu) and barley D-hordein (D-Hor) and B-hordein (B-Hor) storage protein promoters maintained endosperm-specific expression of green fluorescent protein (GFP) in wheat and barley, respectively. Using gfp, we have demonstrated that the Isa and Em promoters can be used as strong promoters to direct transgenes in specific tissues of barley and wheat grain. Differential promoter activity across cereals expands and adds value to a promoter toolbox for utility in plant biotechnology.