Technical Abstract: Post-flowering drought tolerance is an essential trait for increasing the production of sorghum [Sorghum bicolor (L.) Moench] and other cereals in Mediterranean and semiarid tropical climates. Current methodologies for identifying the nonsenescent (stay-green) trait require the right intensity of drought stress at the right developmental stage to visually evaluate lines in the field. Field-based evaluations of drought tolerance are notoriously difficult to manage, and often require growing lines in multiple locations across several years to acquire a meaningful assessment of the stay-green trait. By means of a 30-min high-temperature challenge to leaf tissue during flowering of well-watered sorghum and a 30-min room temperature recovery, we show that stay-green lines can be readily identified. Using chlorophyll fluorescence to monitor tissue injury, we found that tissue with higher intercellular sucrose concentrations exhibited higher chlorophyll fluorescence yield following the temperature challenge. Stay-green lines evaluated in this study maintained higher dawn leaf sucrose levels than the senescent lines among the five youngest leaf positions. Evaluation of 10 known stay-green and senescent sorghum lines, previously reported in the literature, with this bioassay allowed us to separate the two classes of sorghum from well-watered flowering plants. The stay-green lines can also be separated from senescent lines under well-watered greenhouse conditions from the boot stage onward. This technology will greatly reduce the selection time needed to identify drought tolerant sorghum.