An oligo-nucleotide pooled assay (OPA) for high-throughput single nucleotide polymorphism (SNP) genotyping was used for genetic map development in order to coordinate marker information from multiple mapping resources in barley. A doubled haploid (DH) population derived from the cross between barley cultivar "Haruna Nijo" (Hordeum vulgare ssp. vulgare) and wild barley strain "H602" (H. vulgare ssp. spontaneum) was genotyped with 1,448 unigene-derived OPA-SNPs. Of these, 732 markers showed polymorphisms and 384 were cross-referenced with EST markers on our high-density transcript map. The OPA-SNP markers were well distributed on barley chromosomes as follows: 1H (93), 2H (131), 3H (123), 4H (97), 5H (108), 6H (92) and 7H (88). Using a cMAP platform, it was possible to integrate EST marker positions across high-density EST maps. The OPA-SNPs were used to genotype 99 BC(3)F(5) recombinant chromosome substitution lines (RCSLs) from the same cross (Haruna Nijo/H602). These data were used to create graphical genotypes for each line and thus estimate the location, extent, and total number of introgressions from the wild barley parent. The RCSLs sampled most of the wild barley genome, with only a few missing segments. With the resources we have developed, all QTL alleles segregating in this germplasm are now potential targets for map-based cloning.