Nutrient deficiencies are a significant abiotic stress of soybean. Iron deficiency chlorosis is a major concern in the upper midwestern region of the United States due to the prevalence of calcareous soils. Soybean's susceptibility to iron stress results in yield losses into the hundreds of millions each year. Understanding the molecular differences between resistant and susceptible cultivars will significantly affect future yield and revenue. Through the use of near-isogenic lines (NILs), molecular markers, and gene expression we have identified the donor parent introgressions through both classical SSR mapping and a novel method of SNP clustering which can be preformed using data generated through either chip-based SNP genotyping platforms or identified de novo though re-sequencing techniques. By aligning the newly constructed introgression map with the previously identified Fe efficiency QTL we identified a region on chromosome 3 where the two were positionally coincident. To further narrow this region of interest, the NIL was backcrossed an additional generation to the recurrent parent in order to identify recombinations within the chromosome 3 introgression. These lines were identified as Sub-NILs. Recombinants were identified in regular intervals throughout the introgression and phenotyped. Donor parent alleles identified within a 250 kb region represented the minimum interval differentiating the efficient and inefficient Sub-NILs. A second NIL sharing the same donor parent was screened for introgressions. The only region of the genome the two NILs shared alleles from the donor parent, introgressions, were localized to the same region on chromosome 3 further adding support to the importance of the these alleles. Eighteen genes were annotated within the region and were screened for gene expression differences in soybean roots 24 hours following the removal of iron in the growth medium. Two of the genes were differentially expressed between sufficient and insufficient iron conditions. Interestingly, these genes are homologs of two transcription factors in Arabidopsis thaliana known to function in the iron response pathway. Sanger sequencing of these two genes identified a significant mutation that deletes 4 amino acids in the susceptible lines. We hypothesize that this deletion disrupts the FIT / bHLH heterodimer that has been shown to induce known iron acquisition genes.