Quantitative trait loci (QTLs) have been identified for numerous species since the 1990s using populations developed from biparental crosses. The most common methods of validating QTLs are to quantify their effects in additional mapping populations or test near-isogenic lines (NILs) developed from the original mapping population. These approaches to QTL validation fail to adequately examine the effectiveness of a QTL in breeders' populations. We have developed an alternative QTL validation method in which MLs are developed from existing breeding populations segregating for the QTL. Our objective was to validate this method using Fhb1, a major Fusarium head blight [FHB; causal agent Fusarium graminearum (Schwabe)] resistance QTL in wheat (Triticum aestivum L.). Microsatellite markers flanking the QTL region were used to develop 19 QTL-NIL pairs by sampling F3:4 families from 13 different populations. Each pair was tested in a greenhouse point-inoculation experiment and four field FHB resistance screening nurseries. Near-isogenic lines with the Fhb1 resistance allele had significant (P < 0.001) average reductions of 23% for disease severity ratings and 27% for infected kernels in harvested grain. Disease spread for 9 out of 19 total pairs was significantly (P < 0.05) lower in greenhouse point-inoculation experiments when the Fhb1 resistance allele was present. The QTL validation methodology employed in this study should be broadly applicable to other quantitative traits and plant species.