Elite barley breeding lines from the Australian Northern Region Barley Breeding Program were evaluated at the seedling and adult growth stages for resistance to leaf rust (LR) caused by Puccinia hordei. F3:5 lines derived from parental germplasm of different geographic origins were screened in the glasshouse and field spanning four years of trials. The 2009 and 2011 breeding populations (BP1 and BP2) comprised 360 lines and were genotyped with 3,244 polymorphic diversity arrays technology (DArT) markers. The 2012 and 2013 breeding populations (BP3 and BP4) comprised 320 lines genotyped with the DArT GBS array (DArTseq), providing 15,400 high quality polymorphic markers. Association mapping (AM) using the DArT/DArT-seq datasets and phenotypic data from 15 independent LR response assays identified a number of genomic regions associated with resistance. The BP1 and BP2 study detected a total of 15 QTL; 5 QTL co-located with catalogued LR resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, and Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. Markers in common between the DArT and DArTseq datasets enabled integration of mapping results for LR response across the four breeding populations and all QTL detected were visualised on a single map for validation. The adult plant resistance (APR) locus Rph20 was the only region detected in all field environments. Markers and their associated sequences identified in this study will be useful for building QTL combinations involving Rph20, thereby providing stable LR resistance in improved barley cultivars. We will also highlight the advantages of AM using breeding germplasm over traditional bi-parental mapping approaches that underutilise genetic diversity and divert valuable resources into populations of low breeding value.