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The analysis of allelic variation in model plant species and their wild relatives, such as Hordeum vulgare and Hordeum spontaneum, is useful for relating genetic determinants and important phenotypic traits such as stress tolerance. High resolution melting (HRM) analysis is a cost-effective, rapid, and high-throughput assay for mutation screening and genotyping without sequencing. The present study describes an HRM analysis of natural sequence variation within Dhn3 alleles from different H. spontaneum accessions. Small PCR-derived amplicon assays were developed for exon 1 and exon 2 of Dhn3. The efficiency of the HRM procedure was affected by various factors, including specificity and efficiency of PCR, amplicon length and position, and DNA template quality. In addition to these factors, the use of PCR product rather than genomic DNA in HRM increased the quality of melting curves, thus affecting the accuracy and sensitivity of the assay. HRM classified 5-6 groups of variants carrying deletion mutants and single and multiple SNPs consistent with the sequencing data. 18-bp deletion variants were distinguished from the reference sample according to HRM analysis of 207-bp fragment of exon 1. A/T conversions were difficult to discriminate variants, whereas A/G or T/C transitions were easily grouped because they required high T m differences. The conditions and parameters for an optimized HRM assay were outlined, and its potential application in allelic variation research of stress-related genes was discussed.