Non-destructive high-throughput phenotyping based on phenomics is an emerging technology for assessing the genetic diversity of various traits and screening in breeding programmes. In this study, non-destructive measurements of leaf temperature and chlorophyll fluorescence were conducted to investigate the physiological responses of soybean (Glycine max) to salt stress so as to set up a non-destructive screening method. Two-week-old seedlings of soybean in the V2 stage were treated with 0, 12.5, 25, 50 and 100 mM NaCl to induce salt stress. Three parameters, photosynthesis rate, stomatal conductance and chlorophyll fluorescence, decreased significantly, while soybean leaf temperature increased by exhibiting a positive correlation with NaCl concentration (P< 0.001). Soybean leaf temperature increased significantly at 50 mM NaCl when compared with the untreated control, although no visual symptom was observed. We selected leaf temperature as a major physiological parameter of salt stress as its measurement is much easier, faster and cheaper than that of other physiological parameters. Therefore, leaf temperature can be used for evaluating the responses to salt stress in soybean as a non-destructive and phenomic parameter. The results of this study suggest that non-destructive parameters such as chlorophyll fluorescence and leaf temperature are useful tools for assessing the genetic diversity of soybean with regard to salt stress tolerance and to screen salt stress-tolerant soybean for breeding.