WRKY-type transcription factors have multiple roles in the plant defence response and developmental processes. Their roles in the abiotic stress response remain obscure. In this study, 64 GmWRKY genes from soybean were identified, and were found to be differentially expressed under abiotic stresses. Nine GmWRKY proteins were tested for their transcription activation in the yeast assay system, and five showed such ability. In a DNA-binding assay, three proteins (GmWRKY13, GmWRKY27 and GmWRKY54) with a conserved WRKYGQK sequence in their DNA-binding domain could bind to the W-box (TTGAC). However, GmWRKY6 and GmWRKY21, with an altered sequence WRKYGKK, lost the ability to bind to the W-box. The function of three stress-induced genes, GmWRKY13, GmWRKY21 and GmWRKY54, was further investigated using a transgenic approach. GmWRKY21-transgenic Arabidopsis plants were tolerant to cold stress, whereas GmWRKY54 conferred salt and drought tolerance, possibly through the regulation of DREB2A and STZ/Zat10. Transgenic plants over-expressing GmWRKY13 showed increased sensitivity to salt and mannitol stress, but decreased sensitivity to abscisic acid, when compared with wild-type plants. In addition, GmWRKY13-transgenic plants showed an increase in lateral roots. These results indicate that the three GmWRKY genes play differential roles in abiotic stress tolerance, and that GmWRKY13 may function in both lateral root development and the abiotic stress response.