Plant immunity against the majority of microbial pathogens is conveyed by a phenomenon termed non-host resistance (NHR). This multifactorial trait provides durable protection against a given pathogen species. We investigated the molecular basis of NHR in Arabidopsis against the wheat leaf rust pathogen, Puccinia triticina (Ptr). Urediospores germinated with high efficiency and grew randomly over the Arabidopsis leaf surface. However, only 12% of urediospores produced a germ tube that successfully located a stoma and just 0.2% of urediospores went on to produce a haustorium within a penetrated mesophyll cell. Attempted Ptr infection induced the production of reactive oxygen intermediates (ROIs), nitric oxide (NO), salicylic acid (SA) and camalexin. The expression of SA, jasmonic acid (JA) and ROI-dependent genes was also detected. A series of well-characterized defence-related mutants were challenged with Ptr, but none of these lines exhibited significantly increased susceptibility to this fungus. Our findings also suggest that attempted Ptr infection triggers transient stomatal closure in Arabidopsis. We assessed the response of a collection of 79 geographically diverse Arabidopsis accessions to Ptr. Wa-1 plants supported a striking increase in Ptr substomatal vesicle frequency relative to all other tested accessions. Furthermore, SA and camalexin levels became elevated in Wa-1 plants relative to the Col reference line, in response to attempted Ptr infection. Additionally, the kinetics of SA-dependent gene expression was expedited in this accession relative to Col plants. To uncover the genetic architecture of NHR against Ptr, we assayed the response of the Arabidopsis Landsberg erecta (Ler) × Columbia (Col) recombinant inbred population to this fungus. Multiple small-to-medium effect quantitative trait loci were identified that govern the expression of NHR in Arabidopsis against Ptr.