Eddy covariance measurements were made in seven fields in the Midwest USA over 4 years (including the 2012 drought year) to estimate evapotranspiration (ET) of newly established rain-fed cellulosic and grain biofuel crops. Four of the converted fields had been managed as grasslands under the USDA’s Conservation Reserve Program (CRP) for 22 years, and three had been in conventional agriculture (AGR) soybean/corn rotation prior to conversion. In 2009, all sites were planted to no-till soybean except one CRP grassland that was left unchanged as a reference site; in 2010, three of the former CRP sites and the three former AGR sites were planted to annual (corn) and perennial (switchgrass and mixed-prairie) grasslands. The annual ET over the 4 years ranged from 45% to 77% (mean = 60%) of the annual precipitation (848–1063 mm; November–October), with the unconverted CRP grassland having the highest ET (622–706 mm). In the fields converted to annual and perennial crops, the annual ET ranged between 480 and 639 mm despite the large variations in growing-season precipitation and in soil water contents, which had strong effects on regional crop yields. Results suggest that in this humid temperate climate, which represents the US Corn Belt, water use by annual and perennial crops is not greatly different across years with highly variable precipitation and soil water availability. Therefore, large-scale conversion of row crops to perennial biofuel cropping systems may not strongly alter terrestrial water balances.