The electronic structure of the ferrous ion in the Fe(II)-5′-GMP complex has been determined by means of least-squares fittings of the temperature dependence of both the quadrupole splitting and the magnetic susceptibility experimental data, using a crystal-field approximation. The resulting low lying energy levels in order of increasing energy were 5B2, 1A1, 5E, and 3E, but after applying the rhombic perturbation, the rhombic-split 5E term became the ground state due to a quite large rhombic distortion D=−2236 cm−1. The set of crystal-field parameters obtained from the fit has been correlated with the molecular structure of the Fe(II)-5′-GMP derivative. The great value of the ε2 parameter is indicative of a strong N7–Fe interaction while the values of the ε3 parameter and the rhombic distortion are in agreement with a large distortion in the octahedron plane. Finally, the Debye temperature of the solid has been derived from the thermal variation of the Lamb–Mössbauer factor and has been used to calculate the second-order Doppler shift of the ferrous cation which, together with the assumption of a linear temperature dependence for the intrinsic isomer shift, reproduces the isomer shift experimental data.