Soil-borne barley yellow mosaic virus disease - caused by a complex of at least three viruses, i.e. Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus (BaYMV) and BaYMV-2 - is one of the most important diseases of winter barley in Europe. The two genes rym4, effective against BaMMV and BaYMV, and rym5, additionally effective against BaYMV-2, comprise a complex locus on chromosome 3HL, which is of special importance to European barley breeding. To provide the genetic basis for positional cloning of the Rym4/Rym5 locus, two high-resolution maps were constructed based on co-dominant flanking markers (MWG838/Y57c10 - MWG010/Bmac29). Mapping at a resolution of about 0.05% rec., rym4 has been located 1.07% recombination distal of marker MWG838 and 1.21% recombination proximal to marker MWG010. Based on a population size of 3,884 F2 plants (0.013% recombination) the interval harbouring rym5 was delimited to 1.49 ± 0.14% recombination. By testing segmental recombinant inbred lines (RILs) for reaction to the different viruses at a resolution of 0.05% rec. (rym4) and 0.019% rec. (rym5), no segregation concerning the reaction to the different viruses could be observed. AFLP-based marker saturation for rym4, using 932 PstI + 2/MseI + 3 primer combinations only resulted in three markers with the closest one linked at 0.9% recombination to the gene. Two of these markers detected epialleles arising from the differential cytosine methylation of PstI sites. Regarding rym5, profiling of 1,200 RAPD primers (about 18,000 loci) and 2,048 EcoRI + 3/MseI + 3 AFLP primer combinations (about 205,000 loci) resulted in one RAPD marker and seven AFLP markers tightly linked to the resistance gene. Flanking markers with the closest linkage to rym5 (0.05% and 0.88% recombination) were converted into STS markers. These markers provide a starting point for chromosomal walking and may be exploited in marker-assisted selection for virus resistance based on rym5.