Fusarium head blight (FHB) and its associated mycotoxin, deoxynivalenol (DON), are the major biotic factors limiting cereal production in many parts of the world. A recent association mapping (AM) study of US six-row spring barley identified several modest effect quantitative trait loci (QTL) for DON and FHB. To date, few studies have attempted to verify the results of association analyses, particularly for complex traits such as DON and FHB resistance in barley. While AM methods use measures to control for the effects of population structure and multiple testing, false positive associations may still occur. A previous AM study used elite breeding germplasm to identify QTL for FHB and DON. To verify the results of that study, we evaluated the effects of the nine DON QTL using near-isogenic lines (NILs). We created families of contrasting homozygous haplotypes from lines in the original AM populations that were heterozygous for the DON QTL. Seventeen NIL families were evaluated for FHB and DON in three field experiments. Significant differences between contrasting NIL haplotypes were detected for three QTL across environments and/or genetic backgrounds, thereby confirming QTL from the original AM study. Several explanations for those QTL that were not confirmed are discussed, including the effect of genetic background and incomplete sampling of relevant haplotypes.