Technical Abstract: Non-host resistance (NHR) confers plant species immunity against the majority of microbes. As an important crop, wheat can be damaged by several Puccinia species but is immune to all Uromyces species. Here, we studied the basis of NHR in wheat against the broad bean rust pathogen Uromyces fabae (Uf). In the wheat–Uf interaction, microscopic observations showed that urediospores germinated efficiently on wheat leaves. However, over 98% of the germ tubes failed to form appressoria over stomata. For the few that invaded through stomata, the majority of them failed to penetrate wheat mesophyll cells. At 96 hours after inoculation, less than 4% of the Uf infection units that had entered the mesophyll tissue formed haustoria. Attempted penetration by haustorium mother cells induced the thickening of cell wall and the formation of papillae in plant cells, which arrested the development or growth of Uf penetration pegs. For the Uf haustoria formed in wheat cells, they were encased in callose-like materials and did not elicit hypersensitive response. Localized accumulation of H2O2 were observed in plant cell walls, papillae and encasement of haustoria during the wheat–Uf interaction. Furthermore, quantitative RT-PCR analysis showed that several genes involved in basal resistance and oxidative stress responses were up-regulated during Uf infection. In conclusion, our study revealed the cytological and molecular bases of NHR in wheat against the non-adapted rust fungus Uf, and highlighted the significance of papilla production in the prehaustorial NHR.