The aim of this study was to investigate the effect of different sources of iron, hormones and Agrobacterium tumefaciens in the chlorophyll and iron concentration of the peach cultivars Katerina and Fire Blight. The results showed that the concentration of Fe in the leaves was significantly increased after spraying with FeSO₄.7H₂O (alone or in combination with KNO₃, urea, citric acid or ascorbic acid) or with GA₃ + Kinetin. Soil applications with Fe-EDDHA also significantly increased the iron concentration in leaves. Chlorophyll (a/b) increased only in trees sprayed with citric acid or H₂SO₄ 60 days after application. SPAD chlorophyll measurements showed that Fe-EDDHA and FeSO₄.7H₂O + organic matter, applied as soil drench, significantly increased the chlorophyll concentration in leaves of Katerina even up to 120 days after application. Foliar treatments had no significant effect on the SPAD index. SPAD measurements also showed that the concentrations of chlorophyll in leaves of the rootstocks St. Julien 655/2 and wild seedling rootstocks were significantly lower than in GF677 and Antafuel. The rootstocks inoculated with the A. tumefaciens strain Ag-28 alone showed a significantly lower chlorophyll concentration than the uninoculated ones.