This study defines key genes involved in Fe homeostasis and reveals cultivar differences in Fe deficiency responses in peanuts. A comparative transcriptome analysis was conducted on two peanut cultivars, Silihong and Fenghua 1, under Fe-sufficient and -deficient conditions. Plant growth and metal contents were measured. Silihong is more tolerant to Fe-deficiency than Fenghua 1. Fe deficiency increased the uptake of Zn, Mn, Mg, Ca and Cu in peanut plants. A total of 56 and 19 transporter genes were identified to be differentially expressed genes in Silihong and Fenghua 1 respectively. Two cultivars shared twelve common differentially expressed genes encoding OPT3, FRO2, Nramp3, YSL3, IRT1, FER3 and FRO7. Silihong showed higher expressions of AHA4, COPT1, FRO2, IRT1, MTP4, MTP9, Nramp5, OPT3 and VITH4, while Fenghua 1 exhibited higher expressions of ATX1, HMA5, MPT1, OPT6, YSL3, YSL7, ZIP1, ZIP4, ZIP5 and ZIP11 under Fe deficiency. Peanuts overcome Fe deficiency by increasing Fe uptake and altering its transport pathway, in which FRO2, IRT1, Nramp3, YSL3 and OPT3 are involved. Higher expression of AHA4, FRO2, IRT1 and Nramp5 might be responsible for higher Fe-deficiency tolerance in Silihong. Induction of IRT1, Nramp5 and COPT1 by Fe deficiency contribute to increased metal uptake in peanut plants.