Technical Abstract: We previously identified a region near the centromere of barley chromosome 6H that harbors two closely linked net form net blotch resistance genes (rpt.r and rpt.k). In this study, we mined barley EST databases and developed EST-derived markers for further mapping of this region in a population derived from Rika and Kombar. Additionally, we did a comparative analysis of this region to other grass species. Fifteen newly developed markers were added to the previous map, and most were distributed within a 27.0 cM interval spanning rpt.r and rpt.k. The two resistance loci were delineated to a 1.6-cM genetic interval. Comparison of mapped ESTs along chromosome 6H to wheat bin-mapped ESTs suggests that rpt.r and rpt.k are most likely located on the long arm of chromosome 6H. Comparative analysis revealed that a 12.6 cM region containing the two resistance loci is colinear with a region of rice chromosome 2 between 23.7 and 29.9 Mb with only a few rearrangements, and nearly the same level of colinearity was observed between barley and Brachypodium in this region. Ten markers adjacent to the two resistance loci were tested on 20 barley net blotch resistance gene differential lines, and only two and one lines were found to have the same haplotype as Kombar and Rika, respectively, suggesting the other lines may contain different resistance genes. This work refines the genetic and physical location of rpt.r and rpt.k and provides an initial step towards the map-based cloning of these two genes.