The production of cereal crops is essential to secure a future that feeds the continuously growing population. Rust fungi reduce host fitness by feeding on their living tissue and interfere with the global production of crops. Cereal rusts, like Puccinia hordei (the causal agent of barley leaf rust) and Puccinia triticina (the causal agent of wheat leaf rust), have a narrow host range and colonize only one particular species. The most durable type of resistance, non-host resistance (NHR), is the immunity of an entire plant species to all strains of a pathogen species. Exploring the genetics of NHR has proven to be challenging because most interspecific hybrids are infertile. Previously, barley Rphq2 and Rph22, which encode orthologous lectin receptor-like kinases (LecRKs), were transformed into an experimental barley line, Golden SusPtrit, and showed resistance against adapted and non-adapted leaf rust species. We used these transgenic barley lines in the current project to explore the effect of the LecRKs on four wheat leaf rust (P. triticina) isolates. We used the settling tower method to inoculate four isolates of P. triticina on Rphq2 and Rph22 transgenic families. We found that most transgenic families showed an increase in resistance compared to the non-transgenic control 750-E1. By measuring the infection frequency of the infections, we identified that P. triticina isolates 93012 and 95012 had opposite virulence effects on two barley families, Rphq2-E5 and Rph22-E2A. Although the expression levels of Rphq2 and Rph22 followed an induction trend, we did not find significant differences between the isolates. We conclude that resistance mediated by Rphq2 and Rph22 against P. triticina isolates does not involve an isolate-specific component. Thus, we propose investigating differences between rust species to further explore the molecular aspect of non-host resistance.