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Iron chlorosis is one of the most serious abiotic stresses affecting citrus (Citrus sp.) culture in the Mediterranean Basin. A trial was performed with potted tolerant and sensitive rootstocks that were grown in volcanic and calcareous soils. Microarray analysis allowed for the identification of differentially expressed genes putatively involved in iron (Fe) deficiency. Most of the differentially expressed genes isolated from the root tips were of unknown function; the remaining genes were related to the oxidative stress response (e.g., glutathione peroxidase), hormone metabolism and signaling (e.g., small auxin up RNA family protein genes), biological regulation, protein turnover, and the tricarboxylic acid cycle (e.g., aconitase). Additionally, the majority of the Fe stress-related genes expressed in the sensitive Swingle citrumelo (Citrus paradisi × Poncirus trifoliata) and tolerant Carrizo citrange (Citrus sinensis × P. trifoliata) rootstocks identified using real-time reverse transcription–polymerase chain reaction (RT-PCR) were related to regulation, the oxidative stress response, and hormone metabolism and signaling, thereby confirming the array data. Furthermore, validation of the differentially expressed genes in seven tolerant and sensitive rootstocks grown in a field trial under chlorotic conditions was performed. In general, the gene expression profiles reflect the different responses of rootstocks, possibly as a result of the various genetic mechanisms involved in the response to Fe deficiency. Moreover, the expression of aconitase was analyzed in the roots and juice to evaluate the implication of the different aconitase isoforms (Aco), which are derived from specific cellular compartments, in the different tissues. The involvement of the mitochondrial isoform (Aco2) was directly correlated with the acidity of the juice, whereas the cytosolic one (Aco3), which corresponds to the aconitase isolated during the microarray analysis, was found specifically in the roots.