As aboveground biomass of perennial grasses is harvested for feedstock or bioenergy production, root biomass C and N become primary C and N inputs for enhancing soil C and N sequestration. Information is scanty about root biomass C and N and subsequent soil C and N stocks under bioenergy perennial grasses applied with various N fertilization rates in semiarid regions. We evaluated the effect of perennial grass species and N rates on root biomass C and N and soil total C (STC) and total N (STN) stocks at the 0-120 cm depth from 2011 to 2013, 2-4 yr after grass establishment, in the northern Great Plains, USA. Perennial grasses were intermediate wheatgrass (Thinopyrum intermedium [Host] Barkworth and Dewey), smooth bromegrass (Bromus inermis L.), and switchgrass (Panicum virgatum L.), and N fertilization rates were 0, 28, 56, and 84 kg N ha(-1). Root biomass C and N at 0-15 and 0-120 cm were greater with intermediate wheatgrass and switchgrass than smooth bromegrass in 2011, but the trend reversed for root biomass C at 0-15 cm in 2012. Root biomass C at both depths among N rates and years. The STC at 0-15, 30-60, and 0-120 cm also varied among grass species and years. At 30-60 cm, STC increased with increased N rates under intermediate wheatgrass and switchgrass, but decreased under smooth bromegrass. At 60-90 cm, the trend for STC reversed. The STN at 15-30 cm was greater under intermediate wheatgrass than smooth bromegrass and switchgrass and at most depths was greater in 2012 than 2013. Overall root biomass C and N at 0-120 cm were 12-16 times greater and STC and STN 8-9% greater under perennial grasses than adjacent annual spring wheat. Although intermediate wheatgrass returned more root C and N to the soil than other grasses, both STC and STN varied among grass species and N rates. Increased root C and N inputs, however, increased STC and STN under perennial grasses compared with annual spring wheat.