AbstractCitrus established in calcareous soils can be affected by iron (Fe)‐deficiency chlorosis which limits yield and the farmers' income. The degree of deficiency depends on the rootstock, but the resistance to Fe chlorosis still requires further investigation. To study physiological parameters of citrus rootstocks that could be used to evaluate resistance to Fe deficiency, plants of Troyer citrange (Citrus sinensis L. Osb. × Poncitrus trifoliata L. Raf.), Carrizo citrange, Volkamer lemon (Citrus volkameriana Ten. & Pasq.), alemow (Citrus macrophylla Wester), and sour orange (Citrus aurantium L.) were grown in nutrient solutions with 0, 5, 10, 15, or 20 μM Fe. For each rootstock, plant height, root and shoot dry weights, and concentration of Fe in the shoots and roots were measured at the end of the experiment. Chlorophyll (CHL) concentration was estimated throughout the experimental period using a portable CHL meter (SPAD‐502) calibrated for each rootstock. At the end of the experiment, CHL fluorescence parameters were measured in each rootstock with a portable fluorimeter. Maximal and variable fluorescence values indicated that the photochemistry of Troyer was more affected by a low concentration of Fe in the nutrient solution than that of other rootstocks. To compare rootstocks, the absolute CHL concentration was converted into relative yield by employing a scaling divisor based on the maximum value of total CHL in plants without Fe‐deficiency symptoms. Exponential models were developed to determine the minimum Fe concentration in nutrient solution required to maintain leaf CHL at 50% of the maximum CHL concentration (IC50). Models were also developed to assess the period of time the rootstocks were able to grow under Fe‐stress conditions before they reached IC50. Volkamer lemon and sour orange needed the lowest Fe concentration (between 4 and 5 μM Fe) to maintain IC50, and Troyer citrange had the highest Fe requirement (14 μM Fe). Citrus macrophylla and Carrizo citrange required 7 and 9 μM of Fe, respectively. Similarly, Volkamer lemon and sour orange rootstocks withstood more days under total Fe depletion or with a low concentration of Fe (5 μM Fe in nutrient solution) until they reached IC50, compared to the other rootstocks. The approach used led to a classification of the rootstocks into three categories, regarding their internal tolerance to Fe chlorosis: resistance (sour orange and Volkamer lemon), intermediate resistance (C. macrophylla and Carrizo citrange), and reduced resistance (Troyer citrange).