Near infrared spectra obtained from leaf surfaces of Eucalyptus grandis seedlings were calibrated against physiological measurements of plant water stress, namely: relative water content, leaf water potential and stomatal conductance. In a controlled environment facility, spectral data were obtained from both abaxial and adaxial leaf surfaces as well as upper and lower leaves in the stem. The strongest coefficients of determination using cross-validation were r2CV = 0.85 for relative water content, r2CV = 0.74 for leaf water potential and r2CV = 0.80 for stomatal conductance. The use of a portable NIR instrument enabled the rapid assessment of physiological status of seedlings and would enable in situ, non-destructive, high-throughput monitoring of drought and recovery in the field. This would be particularly useful for non-destructive measurement in longitudinal studies.