The high economic losses caused by the occurrence of iron chlorosis in Prunus orchards in the Mediterranean area justifies the implementation of breeding programs to generate high-performance rootstocks for different edaphoclimatic area conditions. For that reason, the genetic control of iron chlorosis tolerance was studied in an F-1 population derived from a three-way interspecific cross between a Myrobalan plum (P 2175) and an almond x peach hybrid (Felinem). Several phenotypic measurements were assessed to guarantee an accurate data set for genetic analysis. SPAD (Soil and Plant Analyzer Development) values, chlorophyll concentration, and visual diagnostic symptoms were highly correlated with leaf chlorosis in trees. SPAD value was the most reliable measure, since it was an objective, unbiased, and non-destructive method. Two significant quantitative trait loci (QTLs) involved in SPAD and chlorophyll concentration were identified for Felinem in linkage groups 4 and 6. Both QTLs were detected in four of the six consecutive years of the experiment. For P 2175, two of the three putative QTLs identified, pspad4.1 and chl4.1, were placed in linkage group 4. These QTLs were related to the SPAD values and chlorophyll concentration, respectively, and co-localized with QTLs detected in the Felinem map affecting the same traits. Candidate gene PFIT, related to iron metabolism, was localized within the confidence interval of the QTL in linkage group 4. This research suggests an association of this chromosome region with tolerance to iron chlorosis in Prunus, and it provides a first approach to localize candidate genes involved in tolerance to this abiotic stress.