The effects of drought on soybean during reproductive development were evaluated in a line-source experiment (2012-2014) that included three water treatments: well-watered (WW), partially-watered (PT), and rainfed (RF). Irrigation was applied in the WW treatment when the estimated soil-water deficit reached 30 mm. Five fast and five slow-wilting genotypes from a population derived from Benning×PI416937 were evaluated in this study. Carbon isotope discrimination (CID) and oxygen isotope composition (OIC) were determined from leaves (CID only) sampled at late R5 and from seed (for CID and OIC) at harvest as a surrogate measure for water use efficiency and transpiration amount, respectively. Additionally, aerial thermal infrared images were taken from balloon or kite platforms. CID values for leaf (2012) and seed (2012, 2013) decreased with increasing intensity of drought stress, while OIC values for seed (2012) increased with increasing intensity of drought stress. Likewise, slow-wilting genotypes had low CID (2013 and 2014 for leaf, 2012 and 2014 for seed) and OIC (2013 for seed) values compared to fast-wilting genotypes regardless of stress intensity, indicating that slow-wilting genotypes had higher water use efficiency and lower transpiration than fast-wilting genotypes. Canopy temperature increased with decreased water availability (2013, 2014), and slow-wilting genotypes had low canopy temperatures compared to fast-wilting genotypes within each of the water treatments (2013). These results indicate that CID and OIC measurements of leaf and seed and canopy temperature measurements hold promise for rapidly characterizing drought-related traits in soybean.