Oat production in the Mediterranean region faces important yield and quality challenges, with crown rust (caused by Puccinia coronata f. sp. avenae) being one of the most important critical biotic constraints. Crown rust populations in the Mediterranean basin are reported to exhibit more virulence compared with those in central and northern Europe. Breeding oat for disease resistance is the most efficient, economic, and environmentally sustainable strategy for managing biotic stresses. However, development of resistant varieties is an ongoing challenge for breeders, as newly released varieties carrying specific resistance genes generate selection pressure on pathogen populations, leading to the rapid emergence of new races capable of overcoming these resistance mechanisms. In this work, a Mediterranean oat collection comprising over 700 Avena sativa and A. byzantina accessions was evaluated to identify durable sources of resistance against a highly virulent Mediterranean rust isolate. Phenotypic evaluations were performed at both the seedling stage under controlled conditions and the adult plant stage across three different field environments. Based on macroscopic assessments, 17 resistant accessions were selected for detailed histological analysis to dissect the cellular responses underlying the resistance observed. Microscopic evaluations revealed a diverse array of cellular responses that hampered fungal development at various stages of the infection process: prior to mesophyll penetration, at the penetration stage, and following mesophyll cell penetration. In the latter, triggering of an early or late hypersensitive response (HR) led to cell death, impeding fungal progression. In a few cases, reduced fungal colony size not associated with HR suggests the presence of posthaustorial resistance mechanisms independent of cell death. Field evaluations indicated an overall increase in resistance at the adult plant stage with A. byzantina accessions displaying a more pronounced increase in resistance compared with A. sativa. The identification of the resistant sources together with the characterization of their cellular resistance responses is of great value for breeding programs aiming to enhance resistance durability by integrating both qualitative and quantitative resistance mechanisms. Moreover, this work may contribute to unravelling the molecular basis of rust resistance in oat.