Technical Abstract: Declining freshwater resources, increasing population, and growing demand for biofuels pose new challenges for agriculture research. To meet these challenges, the concept “Blue Revolution” was proposed to improve water productivity in agriculture--“More Crop per Drop”. Sorghum is the fifth most important grain crop and is becoming increasingly important as a biofuel feedstock due to its superior tolerance to water deficit stress. As such, it is commonly grown under rain-fed conditions in the Southern Plains and other semi-arid regions in the world. Thus, its production is strongly affected by the availability of soil water during the growing season. Enhancing transpiration efficiency (TE), defined as biomass accumulation per unit water transpired, may be an effective approach to increase sorghum yield in arid and semi-arid regions under no or limited irrigation. To identify sorghum accessions that use water efficiently, we have configured a high throughput method to screen for TE based on the integrated biomass accumulation over watered used during early vegetative stage, which was demonstrated to be highly correlated with the TE at later developmental stages. Subsequently, large genetic variations were identified from randomly selected 400 sorghum accessions representing all working groups and geological locations. Unlike the high TE traits identified by 13C discrimination method, which are often associated with low transpiration rates and low biomass production, the TE identified by our method is correlated with high biomass production rather than low transpiration. Gas-exchange analysis of eight selected lines indicated that decreased internal CO2 concentration and enhanced PEP carboxylase activity may contribute to the increase in TE. The sorghum accessions with high TE identified in this study may provide genetic materials for improving TE in sorghum breeding.