Long-term pre-breeding using Hordeum vulgare ssp. spontaneum as a donor of bird cherry-oat aphid resistance has resulted in agronomically improved resistance sources of barley along with easy-to-use molecular markers. Bird cherry-oat aphid (Rhopalosiphum padi L.) is a pest and a virus vector in barley to which there are no bred-resistant cultivars. The present study describes how resistance from Hordeum vulgare ssp. spontaneum has been introgressed in cultivated barley via five successive crosses with the same cultivar Lina (BC) and in parallel with other more modern barley cultivars. Most of the selections for resistance are based on measurements of individual aphid growth in the laboratory. This very slow phenotyping method has been complemented by molecular marker evaluation and application in part of the breeding material. Doubled haploid production in each generation has been crucial for more precise selection of lines with the quantitatively expressed resistance. A field trial of selected "BC3"-generation lines essentially confirmed the laboratory results, so did genotyping of the whole pedigree of parents and selected "BC2" and "BC4" offspring lines. The Infinium iSelect 50 K SNP assay confirmed relationships between lines and discerned several new markers for a resistance QTL on chromosome 2H.