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Basal defense is an important component for mitigating disease at early stages of infection. The ability to phenotype at these early stages can provide valuable insights for both pathogen infection and host defense response. Relative qPCR and fluorescence microscopy approaches were utilized to phenotype barley for seedling resistance to wheat stem rust (<i>Puccinia graminis </i>f. sp. <i>tritici</i>) race MCCFC at multiple time points. The susceptible cultivar Steptoe, resistant cultivars containing only <i>Rpg1</i> (Beacon, Morex, and Chevron), and the resistant line Q21861 containing <i>Rpg1</i> and <i>rpg4/Rpg5</i> stem rust resistance genes were evaluated at 24 hours post inoculation (HPI), 48 HPI, 6 days post inoculation (DPI), and 12 DPI. At 12 DPI the seedlings were evaluated using a Stakman scale. Statistical differences (P < 0.05) were found between cultivars as early as 24 HPI using the qPCR assay and displayed similar hierarchal ordering to microscopy observations. Additionally, the fungal development observed at 24 and 48 HPI was different than what was observed at 12 DPI. At early stages, the susceptible cultivar Steptoe had less fungal DNA (more resistant) than the barley lines containing the <i>Rpg1 </i>and <i>rpg4/Rpg5 </i>resistance genes suggesting potential early pre-haustorial resistance contributions. Temporal variation in resistance ranking suggests the qPCR assay may be a valuable method for dissecting pre- and post- haustorial resistance mechanisms in the barley-wheat stem rust pathosystem.