Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a major threat to wheat (Triticum aestivum) and barley (Hordeum vulgare) production. The emergence of the highly virulent Ug99-lineage stem rust races has stimulated research toward the identification and characterization of rust resistance genes in wheat and barley. Populations were developed to elucidate the inheritance and location of Pgt resistance genes in the common wheat landraces PI 626573 and PI 362698. The resistance present in PI 626573 was shown to be conferred by a single dominant gene (SrWLR) and was mapped to a 1.9 cM region on the long arm of chromosome 2B. This region is known to contain Sr9h which is effective against Ug99. SrWLR provides resistance to Pgt race RKQQC and Sr9h does not, suggesting SrWLR may be a new gene or allele of Sr9. Subsequent work has delimited the SrWLR region to 0.36 cM using a synteny-based approach. QTL analysis of the PI 362698 population using Pgt races identified significant (P < 0.1) resistance QTLs on multiple chromosomes. QTLs identified on chromosome 3B map to a similar location as Sr12 which does not provide resistance to Ug99-lineage races, suggesting a new allele or novel resistance gene. The QTLs identified on chromosomes 2B and 6A are thought to be Sr16 or an allele of Sr28 and Sr8a. Sr57 is known to be present in PI 362698 and is thought to be associated with Pgt QTLs detected on chromosome 7D. QTLs on chromosomes 5A and 5B are in regions where Pgt resistance genes have not been previously identified. Relative qPCR, fluorescence microscopy, and infection type approaches were utilized to phenotype barley for seedling resistance to Pgt race MCCFC at multiple time points. Statistical differences (P < 0.05) were found between accessions at 24 hours post inoculation using qPCR and displayed similar hierarchical ordering to microscopy observations. At early stages, the susceptible cultivar Steptoe had less fungal DNA than barley accessions containing resistance genes suggesting potential pre-haustorial resistance contributions. Temporal variation in resistance ranking suggests the qPCR assay may be valuable for dissecting pre- and post-haustorial resistance mechanisms.