An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species.