Puccinia triticina (Pt), the causal agent of leaf rust evolves through forming new pathotypes that adversely affect the growth and yield of wheat cultivars. Therefore, continued production of resistant varieties through exploring novel sources of resistance in wild relatives which are abundantly found in Iran and the neighbouring regions is a major task in wheat breeding programs. The aim of the present study was to explore 60 wild wheat genotypes selected from the species Triticum monococcum, Aegilops tauschii, Ae. neglecta, Ae. cylindrica, Ae. triuncialis, Ae. umbellulata, Ae. speltoides, Ae. columnaris, Ae. crassa and Ae. ventricosa for resistance to leaf rust. The cultivar ‘Boolani’ and Thatcher near‐isogenic lines were used as controls. Two‐week‐old seedlings were inoculated using 10 Pt pathotypes, and the infection types were recorded. The genotypes were also analysed for polymorphism using six sequence‐tagged sites (STS) and sequence characterized amplified region (SCAR) markers. Forty‐eight genotypes produced high infection types (3+) for two pathotypes, but the remaining genotypes produced low infection types of ‘0; =’ to ‘1+CN’ to all pathotypes. The latter included three accessions of Ae. tauschii, two accessions of each Ae. umbellulata, Ae. columnaris and Triticum monococcum, and one accession from each Ae. triuncialis, Ae. ventricosa and Ae. neglecta. Analysis for STS and SCAR markers suggested several genotypes could carry the genes Lr9, Lr10, Lr19, Lr24, Lr26 and Lr37 or their potential orthologs in addition to unknown resistance genes. In conclusion, the identified resistant genotypes could be further characterized and used in wheat breeding programs for leaf rust resistance.