Sorghum [Sorghum bicolor (L.) Moench] is a widely-grown cereal grain and a dietary staple for more than 500 million people worldwide. Sorghum is grown primarily in arid and semi-arid regions with no or limited irrigation. Enhanced transpiration efficiency (TE), defined as total biomass produced per unit water transpired, could have a large impact on sorghum yield and yield stability. We have developed a mini-lysimetric method that directly measures the whole plant TE in sorghum during the early vegetative stage under greenhouse conditions. TE in sorghum lines can be accurately determined in a 2.2-liter pot by using a high water-holding-capacity potting mix with a single saturated watering at the time of planting. The genetic variation in TE of 11 inbred lines and three hybrids were evaluated under two conditions. Although TE was significantly different between the two experiments, similar genotypic rankings were observed across experiments. Our method offers a high throughput and affordable way to determine whole plant level TE in sorghum. This method should be easily amendable to determine TE in other crops.