Barley leaf rust, caused by Puccinia hordei, is one of the most destructive foliar pathogens of cultivated barley, causing significant yield losses in many regions throughout the world. In this study, 521 seedling-susceptible barley germplasm obtained from diverse breeding material sourced from the Australian Winter Cereals Collection (AWCC), the University of Western Australia (UWA), and international collections from Australia, China, Germany, Spain, and Uruguay were assessed for adult plant resistance (APR) in field nurseries over multiple growing seasons. Lines (213) that consistently showed APR over multiple seasons were screened with PCR-based markers closely linked to Rph20 (bPb0837) and Rph23 (EBmac0603). About 93 % of the lines that were resistant in the field carried one or more uncharacterised APR genes with or without Rph20 and Rph23. There was high variability for APR within specific international germplasm collections. The presence of bPb0837 was strongly correlated with high APR (TR-MR) responses in the field, while lines that were positive for the EBmac0603 allele had intermediate resistance (MRMS). Both EBmac0603 and bPb0837 were present in three German lines (Lenka, Line 17 and Volla) and in the Australian variety Macquarie, all were thus were postulated to carry both Rph20 and Rph23 and had TR-20MR responses in the field. Molecular markers closely linked to Rph20 and Rph23 provide a valuable resource that can be used to assist the incorporation of these genes into new cultivars and identify uncharacterised APR.