Spot blotch, caused by Cochliobolus sativus, is an important disease of barley in the Upper Midwest region of the United States. The resistance of six-rowed malting cultivars like Morex has remained effective for over 40 years and is considered durable. Previous research on Steptoe/Morex (S/M), a 6×6-rowed doubled haploid (DH) population, showed that seedling resistance is controlled by a single gene (Rcs5) on chromosome 1(7H) and adult plant resistance by two quantitative trait loci (QTL): one of the major effect on chromosome 5(1H) explaining 62% of the phenotypic variance and a second of minor effect on chromosome 1(7H) explaining 9% of the phenotypic variance. To corroborate these results in a 2×6-rowed DH population, composite interval mapping (CIM) was performed on Harrington/Morex (H/M). As in the S/M population, a single major gene (presumably Rcs5) on chromosome 1(7H) conferred resistance at the seedling stage. However, at the adult plant stage, the results were markedly different as no chromosome 5(1H) effect whatsoever was detected. Instead, a QTL at or near Rcs5 on chromosome 1(7H) explained nearly all of the phenotypic variance (75%) for disease severity. To determine whether this result might be due to the genetic background of the two-rowed susceptible parent Harrington, we analyzed another DH population that included the same resistance donor (Morex) and another six-rowed susceptible cultivar Dicktoo (D/M). Three QTL conferred seedling resistance in the D/M population: one near Rcs5 on chromosome 1(7H) explaining 30%, a second near the centromere of chromosome 1(7H) explaining 9%, and a third on the short arm of chromosome 3(3H) explaining 19% of the phenotypic variation. As in the H/M population, no chromosome 5(1H) QTL was detected for adult plant resistance in the D/M population. Instead, three QTL on other chromosomes explained most of the variation: one on the short arm of chromosome 3(3H) explaining 36%, a second on the long arm of chromosome 3(3H) explaining 11%, and a third at or near Rcs5 on chromosome 1(7H) explaining 20% of the phenotypic variation. These data demonstrate the complexity of expression of spot blotch resistance in different populations and have important implications in breeding for durable resistance.