Roots help in soil water and nutrient uptake and provide carbon (C) input for soil C sequestration, but information on root biomass of bioenergy perennial grasses is lacking. Root/shoot ratios are used to estimate crop root biomass and C inputs, but the values for perennial grasses are also scanty. We examined root biomass, root/shoot ratios, and soil water contents to a depth of 120 cm after grass harvest in the fall for three bioenergy perennial grasses applied with four nitrogen (N) fertilization rates from 2011 to 2013 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 declined with depth and about 60% of the total biomass was located at 0–15 cm where intermediate wheatgrass and switchgrass had higher biomass than smooth bromegrass in 2011. Shoot biomass was greater in intermediate wheatgrass in 2011 and in switchgrass in 2013 than other grasses and increased with increased N rates. Root/shoot ratio was greater in switchgrass than other grasses at 0–120 cm in 2011, but was greater in smooth bromegrass than switchgrass at 0–60, 0–90, and 0–120 cm in 2012 and 2013. Mean root/shoot ratios across N rates and years were not different among grasses and varied from 1.54 at 0–15 cm to 2.54 at 0–120 cm, which were substantially greater than 0.15 and 0.33, respectively, observed for spring wheat (Triticum aestivum L.). Soil water content increased with depth and was greater under switchgrass than other grasses at 0–120 cm in 2011 and 2013. Water content varied with N rate at various soil depths and years. Root biomass was negatively correlated with soil water content (r = −0.56, P = 0.03, n = 15). Because of greater root and shoot biomass, intermediate wheatgrass reduced soil water content due to increased water uptake and will likely provide more C inputs for soil C sequestration from belowground biomass compared to smooth bromegrass and spring wheat.