Ca3Co4O9 is one of the most promising thermoelectric oxide materials at high temperature. Its structure consists of two misfit layers: the CaOCoOCaO rocksalt-type (RS) layer and the CdI2-type CoO2 layer. In this paper, we reported the synthesis of single phase Ca3Co4−xMxO9 (where M = Fe, Cr, and Ga). Thermoelectric measurements showed the variation of thermoelectric properties depending on the charge state of the substitutional elements. We showed the direct evidence for the location of the substitutional elements in the system using an X-ray absorption spectroscopy technique. The extended X-ray absorption fine structure spectra were fitted with the models. Also, the X-ray absorption near edge structure spectra were compared with the simulated spectra from the first principle calculation. The data analysis show that for Fe and Cr substitution, the Fe and Cr atoms are more likely to be located in the RS layer rather than in the CoO2 as presumed previously. For the Ga case, Ga atoms accommodate both sites; still the majority stays in the RS layer. This is supported by the calculation of the total energy of the system, which showed that the total energy is lower when the substituted elements were located in the RS layer.