Iron Deficiency Chlorosis (IDC) is a disease caused by lack of useable iron in the soil. Symptoms include stunting and interveinal chlorosis of the leaves, eventually leading to yield loss at the end of the season. IDC is particularly important in the upper Midwestern United States because soil conditions favor its development. With the use of next generation sequencing approaches, we characterized soybeans’ short and long-term response to iron stress. Our research takes advantage of two near isogenic lines that are 98% genetically identical but differ in their iron efficiency response. Clark plants are iron efficient, while Isoclark plants are iron inefficient and develop symptoms of IDC under iron stress conditions. Both Clark and Isoclark were grown in hydroponics in a greenhouse for ten days. Plants were grown in one of three treatments: iron sufficient media for ten days, iron deficient media for ten days, or iron sufficient media for eight days followed by transfer to iron deficient media for two days. We collected a total of 48 samples (three treatments x two time points x two tissues (roots and leaves) x four replicates) for RNA-seq analysis. This approach allowed us to identify thousands of genes differentially expressed in response to short and long term iron deficiency in both Clark and Isoclark and the transcription factors regulating their expression. These analyses demonstrate the importance of genes involved in cell cycle, gene silencing, iron acquisition and defense in the soybean iron stress response. In addition, they suggest novel mechanisms for signaling between source and sink tissues.