The use of rootstocks tolerant to iron deficiency represents the best alternative to prevent Fe chlorosis for peach production in calcareous soils. Early detection laboratory screening procedures allow the selection of new Fe-efficient rootstock genotypes. Seventeen Prunus rootstocks were tested for root ferric chelate reductase (FC-R) enzymatic activity, leaf SPAD values, and field performance. Some rootstocks were used as a reference to compare with new Prunus selections. Micropropagated plants were grown in hydroponic culture with half-strength Hoagland's nutrient solution containing 90 μm Fe(III)-EDTA as a control treatment. Plants were transferred to iron-free fresh solutions for 4 days and were thereafter resupplied with 180 μm Fe(III)-EDTA for 1 or 2 days. In vivo FC-R activity was measured in all treatments, i.e., control, Fe-deficient, and 180 μm Fe(III)-EDTA resupplied plants. The FC-R activity after Fe resupply was higher in Fe-efficient genotypes such as AdesotoPVP, FelinemPVP, GF 677, Krymsk 86™, and PAC 9921-07 than in the controls. No induction of FC-R activity was found in other genotypes such as Barrier, Cadaman™-AvimagPVP, PAC 9907-23, and PAC 9908-02. An intermediate response was observed in GarnemPVP, Gisela 5PVP, Krymsk 1PVP, Torinel™-AvifelPVP, VSL-2™, and PAC 9904-01. According to the induction of FC-R activity after Fe resupply, genotypes were classified as tolerant, moderately tolerant, or nontolerant to iron-induced chlorosis. These results were compared with SPAD values of plants grown under controlled conditions and in the nursery. Rootstocks that show high induction of FC-R activity also showed high or very high SPAD values in the field.