This paper describes the isolation of the cDNA encoding a protein previously shown to be indicative of the disease-resistance phenotype mediated by the Yd2 gene in barley (Hordeum vulgare L.). Amino acid sequences of four peptides obtained after isolation of the protein on two-dimensional polyacrylamide gels were completely homologous to sequences occurring within subunit E of barley vacuolar proton-translocating ATPase. Nucleotide sequence data of cloned cDNAs from both Yd2 and non-Yd2 barley varieties showed an amino acid change arising from a single-base-pair polymorphism. This was predicted to result in the shift in isoelectric point used previously to differentiate the protein in Yd2 and non-Yd2 barleys. Earlier work had indicated very close linkage between the gene from which this cDNA is derived, which we have named Ylp, and Yd2, the barley yellow dwarf virus resistance gene. We report here the development of PCR-based assays which discriminate between the two alleles of Ylp and thereby act as valuable predictors of Yd2 for barley breeders and others looking to study this important gene in cereal crops. The validity of each assay was tested with an extensive survey of over 100 barley varieties currently under cultivation in Australia or of importance to Australian barley breeding programmes. Complete agreement was observed between the allele of Ylp detected by the assay and the known Yd2 status of the barleys. A dominant PCR marker for the Yd2-associated allele of Ylp was subsequently developed using an allele-specific primer pair. This fast and economical assay will have broad application in the marker-assisted selection of Yd2-containing lines.