The different Fe2+ lattice sites in iron-rich chlorites have been characterized by Mossbauer spectroscopy and molecular orbital calculations in local density approximation. The Mossbauer measurements were recorded at 77 K within a small velocity range (± 3.5 mm s-1) to provide high energy resolution. Additionally, measurements were recorded in a wider velocity range (± 10.5 mm s-1) at temperatures of 140, 200, and 250 K in an applied field (7 T) parallel to the γ-beam. The zero-field spectra were analyzed with discrete Lorentzian-shaped quadrupole doublets to account for the Fe2+ sites M1, M2, and M3 and with a quadrupole distribution for Fe3+ sites. Such a procedure is justified by the results obtained from MO calculations, which reveal that different anion (OH-) distributions in the first coordination sphere of M1, M2, and M3 positions have more influence on the Fe2+ quadrupole splitting than cationic disorder. The spectra recorded in applied field were analyzed in the spin-Hamiltonian approximation, yielding a negative sign for the electric field gradient (efg) of Fe2+ in the M1, M2, and M3 positions. The results of the MO calculations are in quantitative agreement with experimental and reveal that differences in the quadrupole splittings (ΔE(Q)), their temperature dependence and in the isomer shifts (δ) of Fe2+ in M1, M2, and M3 positions can theoretically by justified. Therefore, the combined Mossbauer and MO investigation shows that the three Fe2+ lattice sites in the chlorites investigated here can be discriminated according to their ΔE(Q)-δ parameter pairs. With the calculated average iron-oxygen bond strength, the MO study provides an explanation for the observed trend that the population of the three lattice sites by Fe2+ increases according to the relation M1 < M2 < M3.