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Vast collections of barley germplasm have been established and conserved in various global gene banks. These collections hold tremendous genetic diversity for resistance genes to Puccinia hordei, a causal agent of barley leaf rust. This study was undertaken to discover, characterize and postulate the known Rph genes (resistance to Puccinia hordei) and identify novel sources of ASR (all-stage resistance) and APR (adult plant resistance) to P. hordei. A core set of 315 barley lines were rust-tested as seedlings for their response to eight Australian pathotypes of P. hordei and genotyped with molecular markers linked to the known characterised ASR and APR genes. These tests led to the postulation of ASR leaf rust resistance genes Rph1, Rph2, Rph3, Rph9.am, Rph12, Rph15, Rph19 and Rph25 singly or in combination. Field tests revealed that the vast majority of lines (84%) carried APR. Genotyping of the APR-carrying lines with markers bPb-0837, Ebmac0603 and sun43-44 identified lines that likely carry the known APR genes Rph20, Rph23 and Rph24 singly or in combination. Thirty-nine per cent of the lines were negative for all the three markers and were thus postulated to carry uncharacterized APR. The sources of resistance identified in this study provide a valuable resource to breeders for further utilization and diversifying the genetic basis of leaf rust resistance in barley.