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Iron (Fe) is a mineral micronutrient for plants, and Fe deficiency is a major abiotic stress in crop production because of its low solubility under aerobic and alkaline conditions. In this study, 18 maize inbred lines were used to preliminarily illustrate the physiological mechanism underlying Fe deficiency tolerance. Then biparental linkage analysis was performed to identify the quantitative trait loci (QTLs) and candidate genes associated with Fe deficiency tolerance using the recombinant inbred line (RIL) population derived from the most Fe-efficient (Ye478) and Fe-inefficient (Wu312) inbred lines. A total of 24 QTLs was identified under different Fe nutritional status in the Ye478 × Wu312 RIL population, explaining 6.1–26.6% of phenotypic variation, and ten candidate genes were identified. Plants have evolved two distinct mechanisms to solubilize and transport Fe to acclimate to Fe deficiency, including reduction-based strategy (strategy I) and chelation-based strategy (strategy II), and maize uses strategy II. However, not only genes involved in Fe homeostasis verified in strategy II plants (strategy II genes), which included ZmYS1, ZmYS3, and ZmTOM2, but also several genes associated with Fe homeostasis in strategy I plants (strategy I genes) were identified, including ZmFIT, ZmPYE, ZmILR3, ZmBTS, and ZmEIN2. Furthermore, strategy II gene ZmYS1 and strategy I gene ZmBTS were significantly upregulated in the Fe-deficient roots and shoots of maize inbred lines, and responded to Fe deficiency more in shoots than in roots. Under Fe deficiency, greater upregulations of ZmYS1 and ZmBTS were observed in Fe-efficient parent Ye478, not in Fe-inefficient parent Wu312. Beyond that, ZmEIN2 and ZmILR3, were found to be Fe deficiency-inducible in the shoots. These findings indicate that these candidate genes may be associated with Fe deficiency tolerance in maize. This study demonstrates the use of natural variation to identify important Fe deficiency-regulated genes and provides further insights for understanding the response to Fe deficiency stress in maize.