Technical Abstract: Post-flowering drought tolerance is an essential trait for increasing cereal production in Mediterranean 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 in order to acquire a meaningful assessment of the stay-green trait. By means of a 30-minute high temperature challenge to leaf tissue from pre-flowering well-waterd sorghum and 30-minute 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 ten known stay-green and senescent sorghum lines with this bioassay allowed us to separate the two classes of sorghum from well-watered pre-flowering plants. This technology will greatly reduce the selection time needed to identify drought tolerant sorghum.