Leaf rust (caused by Puccinia triticiana Eriks.) is a wide spread disease of wheat, causing annual yield losses. Here we report, development of KASPar assays for high throughput genotyping of leaf rust resistance locus Lr21. The use of KASP assay in MAS provides a great advantage as large number of individuals could be genotyped in a shorter time, with greater precision and at lower cost. The sequence information of a 4kb region covering the Lr21gene from -92 to +4,261 from 77 cultivars was kindly provided by Dr. Li Huang of Montana State University. A SNP specific to the resistant haplotype at position 2175bp in the NBS region was targeted for designing the KASPar assay. To confirm the efficiency of assay, the SNP marker was applied to DNA of 384 US wheat lines. Although the SNP was specific to lines having Lr21 among the original 77 cultivars, the KASP assay gave ambiguous results (both alleles) in non-Lr21soft winter wheat lines, Application of a Lr21 SCAR marker to these lines revealed the presence of a new, distinguishable haplotype. In order to develop an authentic assay, resequencing of 2.4kb fragment covering the functional region of Lr21 gene in winter wheat lines was done. Alignment of the generated sequence data of this region revealed the presence of a previously reported indel (88 or 105bp) in the first intron of the Lr21 gene in the selected cultivars. A new KASPar assay was designed around a polymorphic SNP (C/T) at position 1353bp and validated in a broad range of germplasm.