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Fusarium head blight (FHB) is a serious disease of cereal grains caused by the fungal pathogen Fusarium graminearum. Deoxynivalenol (or DON), the associated trichothecene mycotoxin is of special concern to barley producers and consumers. A recent association mapping (AM) study of U.S. 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 FHB and DON resistance in barley. Despite control measures used to mitigate the effects of population structure and multiple testing in AM, false positives may still occur. To verify previously reported associations we evaluated the effects of nine DON QTL using near isogenic lines (NILs) for each QTL region. Families of contrasting homozygous haplotypes for each region were derived from lines in the original AM populations that were heterozygous for 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.