Eighty-two Australian and five exotic barley cultivars were evaluated at the seedling stage for resistance to the Australian stem rust pathotype 98-1,2,3,5,6. Although most of these cultivars exhibited mesothetic (mixed infection type) reactions that were associated with a high level of chlorosis, two (‘O'Connor’ and ‘Pacific Ranger’) were highly resistant. Marker analysis indicated that four Australian cultivars (‘Empress’, ‘Vlamingh’, Pacific Ranger, and ‘Yerong’) possess the stem rust resistance gene Rpg1. Tests conducted using North American Puccinia graminis f. sp. tritici pathotypes MCCJ and QCCJ supported marker results and indicated that ‘Pacific Ranger’ and ‘Vlamingh’ likely carry additional stem rust resistance genes. Based on pedigree information and results from multipathotype tests, these genes are believed to be uncharacterized and, therefore, new. The resistance in Australian barley ‘Franklin’ conferred resistance against all pathotypes tested in this study. Studies of inheritance to MCCJ revealed that it possessed an unknown seedling resistance, which was independent of and displayed additivity to Rpg1.