We report the first structural evidence, obtained by synchrotron radiation single-crystal X-ray diffraction experiments, of the presence of preferential substitution sites for iron heteroatoms hosted in the MFI framework of a Na-Fe-silicalite sample. The location of Fe-III at sites T(9) and T(10) is inferred on the basis of (i) an increase of the average T-O distance greater than 3 sigma; (ii) the presence of two peaks (2.28 and 0.67 e/Angstrom (3)) in the electron density map near T(9) and T(10) in a niche of the sinusoidal channel, interpreted as the corresponding Na+ counterion coordinated to framework oxygens; (iii) the higher anisotropy of the thermal displacement parameters observed for T(9) and T(10), which is related to the substitutional disorder of iron insertion. Independent data obtained on single crystals of Fe-silicalite samples synthesized with NH4+ and K+ counterions confirm the results obtained on the sodium derivative.