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Wheat leaf rust caused by Puccinia triticina Eriks is an important disease that causes yield losses of up to 40% in susceptible varieties. Tetraploid emmer wheat (T. turgidum ssp. Dicoccum), commonly called Khapli wheat in India, is known to have evolved from wild emmer (Triticum turgidum var. dicoccoides), and harbors a good number of leaf rust resistance genes. In the present study, we are reporting on the screening of one hundred and twenty-three dicoccum wheat germplasm accessions against the leaf rust pathotype 77-5. Among these, an average of 45.50% of the germplasms were resistant, 46.74% were susceptible, and 8.53% had mesothetic reactions. Further, selected germplasm lines with accession numbers IC138898, IC47022, IC535116, IC535133, IC535139, IC551396, and IC534144 showed high level of resistance against the eighteen prevalent pathotypes. The infection type varied from “;”, “;N”, “;N1” to “;NC”. PCR-based analysis of the resistant dicoccum lines with SSR marker gwm508 linked to the Lr53 gene, a leaf rust resistance gene effective against all the prevalent pathotypes of leaf rust in India and identified from a T. turgidum var. dicoccoides germplasm, indicated that Lr53 is not present in the selected accessions. Moreover, we have also generated 35K SNP genotyping data of seven lines and the susceptible control, Mandsaur Local, to study their relationships. The GDIRT tool based on homozygous genotypic differences revealed that the seven genotypes are unique to each other and may carry different resistance genes for leaf rust.