One of the major concerns with Fusarium head blight (FHB) of barley is the potential health risks to livestock and humans through the accumulation of the mycotoxin deoxynivalenol (DON) in infected grain. To define the role of the host in DON accumulation during the early stages of disease development, we conducted a series of greenhouse experiments. We inoculated single spikelets of greenhouse-grown plants with Fusarium graminearum, moved the plants to a dew chamber, and harvested the inoculated spikelets after 72 h for DON analysis. We conducted a quantitative trait locus (QTL) analysis using a genetic mapping population, constructed with the parents Stander and Frederickson, that segregated for DON accumulation after single-spikelet inoculation in two experiments. A single QTL on chromosome 3 explained 18 and 35% of the phenotypic variation in the two experiments. To validate this QTL for DON accumulation, we used a DNA marker to select near-isogenic lines from a family from the mapping population that was segregating at this QTL. Disease symptom development was similar between the near-isogenic lines; however, the mean DON concentration of the lines homozygous for the allele from the high DON parent was 2.5-fold more than the lines homozygous for the alternate allele. A time course experiment showed that this effect on toxin accumulation was observed at 10 days post inoculation. The near-isogenic lines developed in this study should prove useful for further exploration of the role of DON in FHB.