The conversion of land from existing uses to biofuel cultivation is expected to increase given concerns about the sustainability of fossil fuel supplies. Nonetheless, research into the environmental impacts of biofuel crops, primarily the hydrological impacts of their cultivation, is in its infancy. To investigate such issues, the response of a 1,649 km2 semiarid basin to the incremental substitution of the widely discussed biofuel candidate switchgrass (Panicum virgatum L.) for native land uses was modeled using the Soil and Water Assessment Tool (SWAT). Median discharges decreased by 5.6–20.6% during the spring and by 6.4–31.2% during the summer, depending on the quantity of acreage converted. These were driven by an increased spring and summer evapotranspiration of 3.4–32.0% and 1.5–18.9%, respectively, depending on the quantity of switchgrass biomass produced. The substitution of switchgrass also resulted in larger quantities of water stress days than in baseline scenarios. The authors encourage the exploration of alternative biofuel crops in semiarid areas to mitigate such negative impacts.