The Dehydration Responsive Element-Binding protein/C-repeat Binding Factor (DREB/CBF) plays important roles in regulating physiological processes and downstream gene expression for drought stress in Arabidopsis thaliana, rice, and wheat. 'Essex' soybean [Glycine max (L.) Merr.] was identified as tolerant and a wild soybean PI 407155 (Glycine soja Sieb. & Zucc.) as more tolerant to dehydration stress in a greenhouse screen. In this study, we cloned and characterized the Glycine DREB1 (GlyDREB1) from the dehydration-induced PI 407155 and Essex using a semi-quantitative RT-PCR, rapid amplification of cDNA ends (RACE), and northern blot. We also investigated the physiological characteristics including biomass accumulation, moisture content, and electrolyte leakage for both soy-bean genotypes under dehydration stress. Analysis of homologous GlyDREB1 genes from PI 407155 and Essex indicated differences in both the timing and strength of expression under dehydration stress. The GlyDREB1 in PI 407155 was rapidly induced in 1 h of dehydration shock and the transcription level was much higher than that in Essex. The root system of PI 407155 maintained higher moisture content and biomass accumulation than that of Essex on the 15 d without irrigation. The percentage electrolyte leakage in Essex was almost twice that in PI 407155 under 5 d no irrigation treatment. Both molecular and physiological studies indicated that PI 407155 had higher level of tolerance to dehydration stress than Essex, therefore providing a new potential soybean genome for developing dehydration stress cultivars.