Two-component systems (TCSs) play vital functions in the adaptation of plants to environmental stresses. To identify soybean TCS genes involved in the regulation of drought stress response, we performed tissue-specific expression profiling of all 83 putative TCS genes in plants subjected to dehydration. Under well-watered conditions, the majority of soybean TCS genes were expressed higher in the root tissues. Additionally, a high variability in transcript abundance was observed for the TCS genes in both roots and shoots. Under dehydration, TCS genes were more responsive in shoots than in roots. Further analysis indicated that 50% more TCS genes were repressed by dehydration than induced. Specifically, 18 genes were induced by 2-fold or more, whereas 33 genes were down-regulated at least 2-fold by dehydration. TCS genes putatively involved in cytokinin and ethylene signallings strongly responded to dehydration, suggesting that crosstalk exists between different hormonal and stress pathways. Our study provides the first glance into the complex regulatory roles of soybean TCSs underlying their functions in response to dehydration. Additionally, these systematic expression analyses identified excellent dehydration-responsive candidate genes to further clarify soybean TCS functions in drought response and to enable the development of improved drought tolerance in transgenic soybeans.