Allelic variations at the Glu-B3 (low molecular weight glutenin) locus have been reported to play an important role in determining dough properties and wheat bread-making quality. Due to the complexity of Glu-B3, the effects of different alleles at this locus are not yet fully understood. We evaluated 96 cultivars and elite lines, mainly from the U. S. Great Plains in two dryland locations (Akron and Dailey) and one irrigated location (Fort Collins) in Colorado in the 2005-06 season. Gluten strength and dough mixing properties were measured by 10g mixograph. Three parameters, midline peak volume (MPV), midline right volume (MRV) and midline peak time (MPT) were used as indicators of mixing time and dough strength. Population structure was estimated on the basis of 60 unlinked simple sequence repeat marker loci. The association of allelic variation and mixograph traits was analyzed with a linear regression model with Glu-B3 and subpopulations as fixed factors. The results indicated that growing environments and Glu-B3 allelic variation significantly influenced mixograph traits of these entries. In all three environments, Glu-B3f and h were consistently associated with high MPV and MRV, whereas Glu-B3a, and c were associated with low MPV and MRV; Glu-B3a, b, e and h had consistently high MPT, whereas Glu-B3i and c had consistently low MPT. These results suggest that selection of an appropriate allele at Glu-B3 locus can facilitate the development of cultivars with desirable dough properties for producing different end products.