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<i>Thinopyrum intermedium</i>, commonly known as intermediate wheatgrass (IWG) is a perennial crop shown to have both environmental and nutritional benefits. In comparison to wheat controls, IWG has higher protein and dietary fiber contents. However, a deficiency in high molecular weight glutenins (HMWG), an important component responsible for dough strength, was seen for all IWG lines. Therefore, the objective of this study was to understand the protein distribution of IWG lines and its relationship to bread-making quality parameters. Seventeen IWG lines and two wheat controls were analyzed for molecular weight distribution of flour proteins using size exclusion chromatography and subsequent evaluation of bread-making quality. MALDI-TOF and liquid chromatography coupled with tandem mass spectrometry were performed on extracted gluten from IWG sample and hard red winter wheat to determine molecular weight and identify the HMWG subunits. Dough rheological measurements were performed using farinograph and Kieffer following standard procedures. In contrast to wheat controls, IWG samples had lower percentage of SDS-unextractable high molecular weight polymeric proteins (uHMWPP). This fraction of proteins in IWG lines showed a significant and a positive correlation with farinograph stability and Kieffer-resistance to extension, but lower than that of wheat controls. Relative quantity of soluble albumins and globulins were higher in IWG samples than that of wheat. These findings suggest that lower uHMWPP content in IWG samples results in poor dough rheology due to lack of proper gluten network formation. The difference in protein distribution coupled with higher fiber content in IWG may negatively affect the end-product quality. Investigating the effect of fiber content on dough rheology may help identify ways to utilize IWG grain in commercial applications.