Variation of polymeric proteins affects wheat end-use quality. This research investigated associations of polymeric proteins with dough mixing strength and breadmaking characteristics in a near-homogenous population of 139 recombinant inbred lines (RILs) derived from a cross between two hard red spring wheat breeding lines. Flours from the RILs grown at three locations were analyzed for molecular weight (MW) distribution of SDS-extractable and unextractable proteins using size-exclusion HPLC protocol. Correlations were calculated between mixing and breadmaking properties and HPLC absorbance data obtained a 0.01-min retention time interval to identify protein fractions that had a significant effect on the quality traits. Very high MW polymeric proteins in the unextractable fraction had more distinct and positive associations with dough mixing strength and bread loaf volume than did other polymeric protein fractions, whereas extractable polymeric had negative influence. Consequently, the ratio of unextractable very high MW polymeric proteins to extractable polymeric proteins had greater correlations with dough mixing parameters than other HPLC absorbance area data. Covariate-effect biplots also visually validated positive effects of unextractable very high MW polymeric proteins and negative effects of extractable polymeric proteins on mixing properties and loaf volume across three growing locations.