Genetic resistance is the preferred method to reduce yield losses caused by many diseases including wheat leaf rust. However, in many cases host resistance seems to be short-lived. For decades, scientists have tried and continue trying to understand genetics and physical mechanisms involved in durable host resistance. Incompatible host-pathogen interactions in wheat- Puccinia triticina (Pt) pathosystem can be classified into pre-and post-haustorial. Post-haustorial resistance tends to be involved in race specific resistance and is commonly characterized by presence of hypersensitive reaction (HR), while pre-haustorial resistance do not generally involve HR. The objective of this work was to understand the physical and molecular components associated with resistance mechanisms to Pt. A set of Thatcher near isogenic lines (NIL) carrying different leaf rust (Lr) resistance genes and barley (non-host) genotypes were evaluated in time course experiments for histological analysis and relative fungal DNA quantification. Histological evaluation and q-PCR assay showed differences in time of fungal structures formation and amount of fungal DNA among genotypes. The q-PCR assay could differentiate between resistant and susceptible genotypes at 24 hpi. Additionally, Cq ratio of q-PCR assay allowed for classification of resistant genotypes carrying pre-haustorial (Lr9, Q21860) and those carrying post-haustorial resistance (Lr21, Lr34 and Harrington). To our surprise, despite carrying pre-haustorial resistance, Tc-Lr9 presented HR as early as 6 hpi. Tc-Lr21 presented HR, before (5 hpi) and after haustoria formation (24 hpi). To better characterize the HR and determine if the HR observed was the product of H2O2 accumulation, histochemical and gene expression studies were used. Both methods confirmed that the HR observed in Tc-Lr9 and Tc-Lr21 indeed involved H2O2 accumulation. In addition, up-regulation of hypersensitive induced resistance genes TaHIR1, TaHIR2 was observed in Tc-Lr9. Up-regulation of Tc-Lr21 gene at 5 hpi was observed in Tc-Lr21. The protocols developed and data obtained from this study provide opportunities for quantitatively assess components of resistance and suggest that some previous assumptions about plant-pathogen interaction in host and non-host systems should be revisited.