() is a damaging pathogen of barley throughout the world. Despite its importance, almost nothing is known about the genomics of this pathogen, and a reference genome is lacking. In this study, the first reference genome was assembled for an Australian isolate of ("560") using long-read SMRT sequencing. A total of 838 contigs were assembled, with a total size of 207 Mbp. This included both haplotype collapsed and separated regions, consistent with an estimated haploid genome size of about 150Mbp. An annotation pipeline that combined RNA-Seq of infected host tissues and homology to proteins from four other species predicted 25,543 gene models of which 1,450 genes were classified as encoding secreted proteins based on the prediction of a signal peptide and no transmembrane domain. Genome resequencing using short-read technology was conducted for four additional Australian strains, 612, 626, 608 and 584, which are considered to be simple mutational derivatives of 560 with added virulence to one or two of three barley leaf rust resistance genes (, and ). To identify candidate genes for the corresponding avirulence genes , and , genetic variation in predicted secreted protein genes between the strains was correlated to the virulence profiles of each, identifying 35, 29 and 46 candidates for and , respectively. The identification of these candidate genes provides a strong foundation for future efforts to isolate these three avirulence genes, investigate their biological properties, and develop new diagnostic tests for monitoring pathogen virulence.