Effect of Co substitution in pyrite was investigated using a Raman microprobe. Textural appearance of Co-bearing pyrite was visualized by mapping method. The revealed Raman map tightly correlates with a Co distribution map obtained by electron microprobe and µ-energy-dispersive X-ray fluorescence microscope. In addition, a strong influence on the pyrite spectra due to sample preparation was documented. The standard mechanical polishing caused highly broadened modes at upshifted frequencies, which could be avoided by analyzing of sample polished with Ar broad ion beam or non-polished cut sample. However, the effect of Co on pyrite spectra is independent of the sample preparation. Additionally, the Raman method has several advantages over other methods. For instance, It does not require sample preparation, vacuum chamber and wavelength-dispersive system and operates with a laser instead of X-ray. Thus, the Raman method can be used as a possible tool for differentiation of Co-bearing pyrite from pure one.