The aim of this study was to analyze sulfur content, protein size distribution, and free amino acids in flour mill streams (FMS) and their associations to dough rheology and breadmaking traits. Break FMS had higher nitrogen and sulfur quantities than reduction FMS. The third break FMS had the highest nitrogen and sulfur contents among FMS but low bread loaf volume partly due to high ash content. Sulfur quantity had greater or equivalent correlations with dough rheology and breadmaking properties compared with nitrogen quantity when the effect of percent ash content was removed statistically. FMS also showed significant quantitative variation in HMW polymeric proteins of the SDS-unextractable fraction that had greater association with sulfur content and dough rheology and breadmaking traits than other protein fractions. Asparagine, which is a major amino acid in flour, was found at higher levels in the third break and third reduction FMS. Ratio of nitrogen to sulfur was significantly correlated with asparagine concentration (r = 0.73, α = 0.01). This study indicates that information on sulfur, protein size distribution, and free amino acid is potentially useful in research for more precise blending of FMS in commercial flour mills to meet customer specifications for high quality flour.