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Leaf rust caused by Puccinia triticina Eriks. is a serious threat to wheat production worldwide. Exploiting wild relatives can effectively help to improve the genetic resistance of wheat against rust. This study aimed to evaluate the responses of 34 Aegilops triuncialis accessions to seven Pt pathotypes and to analyse expression of genes associated with resistance at the seedling stage. Following multipathotype testing, two accessions with contrasting resistance were analysed at 0, 6, 12 and 24 h post‐inoculation for the expression profile of genes related to PR1, PR2, PR4, PR9, phenylalanine ammonia‐lyase and ATP‐binding cassette transporter. Of the accessions tested, eight showed high infection types of "3" to at least one Pt pathotype, while the remaining accessions had low ITs ("0;=" to "2+") against all pathotypes. The relative expressions of all candidate genes increased at all‐time points, but the expression level in the incompatible interaction was significantly higher than that of the compatible. The close relationship between PR proteins, PAL, salicylic acid‐ and jasmonic acid‐responsive proteins, as well as the proteins involved in the production of reactive oxygen species and lignin, could point to the specific recognition of the pathogen followed by the timely and intense induction of the expression of PR genes as the main reasons for resistance in incompatible interaction. In conclusion, the present study identified potentially new sources of resistance in this Ae. triuncialis population, which can be exploited in wheat breeding programs. [ABSTRACT FROM AUTHOR]