Phalaris aquatica is a cool-season perennial grass species that is extensively cultivated in Australia, with additional usage in other areas of the world. Phalaris displays a number of desirable agronomic characteristics, although unfavourable traits include excessive seed shattering, sensitivity to aluminium toxicity, and several toxicosis syndromes. Varietal development has to date been based on traditional selection methods, but would benefit from the application of genomics-based approaches, which require the development of large-scale sequence resources. Due to a large nuclear DNA content, methods that target the expressed component of the genome and reduce the complexity of analysis are most amenable to current sequencing technologies. A reference unigene set has been developed by transcriptome sequencing of multiple tissues from a single plant belonging to the variety Landmaster. Comparisons have been made to gene complements from related species, as well as reference protein databases, and patterns of gene expression in different tissues have been evaluated. A number of candidate genes relevant to removal of undesirable attributes have been identified. The reference unigene set will provide the basis for detailed studies of differential gene expression and identification of candidate genes for potential transgenic deployment, as well as a critical resource for genotypic analysis to support future genomics-assisted breeding activities for phalaris improvement.