High intrinsic water-use efficiency (WUEi), the ratio of leaf photosynthesis to stomatal conductance, may be a useful trait in adapting crops to water-limited environments. In soybean, cultivar differences in stomatal response to vapor pressure deficit have not consistently translated into differences in WUEi in the field. In this study, six cultivars of soybeans previously shown to differ in WUEi in indoor experiments were grown in the field in Beltsville, Maryland, and tested for mid-day WUEi on nine clear days during the mid-seasons of two years. Measurement dates were chosen for diverse temperatures, and air temperatures ranged from 21 to 34 °C on the different dates. Air saturation deficits for water vapor ranged from 0.9 to 2.2 kPa. Corrected carbon isotope delta values for 13C (CID) were determined on mature, upper canopy leaves harvested during early pod filling each year. WUEi differed among cultivars in both years and the differences were consistent across measurement dates. Correlations between mean WUEi and CID were not significant in either year. It is concluded that consistent cultivar differences in WUEi exist in these soybean cultivars under field conditions, but that carbon isotope ratios may not be useful in identifying them because of cultivar differences in mesophyll conductance.