Technical Abstract: The glutenin loci of wheat (Triticum aestivum L.) are important determinants of bread-making quality, although the effects of alleles at those loci are incompletely understood. We applied an association analysis method to assess the effects of glutenin alleles and 1RS wheat–rye (Secale cereale L.) translocations on dough-mixing properties in 96 wheat cultivars and advanced lines grown at three Colorado locations while accounting for population structure and relatedness of individuals in the population. The results indicated that (1) in the majority of cases, controlling relatedness of individuals reduced the significance of associations between glutenin loci and Mixograph traits; (2) the Glu-D1 and Glu-B3 loci and 1RS translocations had greater impacts on dough-mixing properties compared to other glutenin loci; (3) Glu-B1w, Glu-D1d, and Glu-B3b were consistently associated with greater (more favorable) Mixograph peak time (MPT) than other alleles at the respective loci, whereas Glu-B1e, Glu-D1a, and Glu-B3c were associated with reduced MPT; (4) the 1BL.1RS translocation was associated with a decrease in Mixograph properties. Our results indicate that taking multiple-level relatedness of individuals into account can improve the results of association analysis for wheat-quality traits.