Abstract Background Barley is an important cereal crop cultivated for malt and ruminant feed and in certain regions it is used for human consumption. It is vulnerable to numerous foliar diseases including barley leaf rust caused by the pathogen Puccinia hordei . Results A temporarily designated resistance locus RphCantala ( RphC ) identified in the Australian Hordeum vulgare L. cultivar ‘Cantala’ displayed an intermediate to low infection type (“;12 = N”) against the P. hordei pathotype 253P- (virulent on Rph1, Rph2, Rph4, Rph6, Rph8 and RphQ) . Phenotypic assessment of a ‘CI 9214’ (susceptible) x ‘Stirling’ ( RphC ) (CI 9214/Stirling) doubled haploid (DH) population at the seedling stage using P. hordei pathotype 253P-, confirmed that RphC was monogenically inherited. Marker-trait association analysis of RphC in the CI 9214/Stirling DH population using 4,500 DArT-seq markers identified a highly significant (−log 10 Pvalue > 17) single peak on the long arm of chromosome 5H (5HL). Further tests of allelism determined that RphC was genetically independent of Rph3, Rph7 , Rph11, Rph13 and Rph14, and was an allele of Rph12 ( Rph9.z ), which also maps to 5HL. Conclusion Multipathotype tests and subsequent pedigree analysis determined that 14 related Australian barley varieties (including ‘Stirling’ and ‘Cantala’) carry RphC and that the likely source of this resistance is via a Czechoslovakian landrace LV-Kvasice-NA-Morave transferred through common ancestral cultivars ‘Hanna’ and ‘Abed Binder’. RphC is an allele of Rph12 ( Rph9.z ) and is therefore designated Rph9.am . Bioinformatic analysis using sequence arrays from DArT-seq markers in linkage disequilibrium with Rph9.am identified possible candidates for further gene cloning efforts and marker development at the Rph9 / Rph12 / Rph9.am locus.