The present work investigates the influence of the preparation of metallographic samples produced for electron backscatter diffraction (EBSD) measurements of near eutectoid steels. Furthermore, the possibility of pearlite colony measurement by means of EBSD and light microscopy is studied. Preparation methods such as, vibratory polishing, electropolishing, preparation with oxide polishing suspension (OPS), ion milling and their combinations were compared. The requirements on the sample surface are essential for EBSD measurements, because the information arises from a depth in the range of only a few nanometers. Moreover, the preparation of pearlitic steels is challenging due to their multiphase structure. The present investigation shows that the preparation with OPS or ion milling yields very good results regarding the indexing rate of EBSD measurements. The highest indexing rate was achieved by combining a preparation with OPS followed by ion milling. For the measurement of the pearlite colonies by means of EBSD, the choice of the correct misorientation angle is decisive. Once the angle is determined, the measurement of the pearlite colony size can be carried out with the evaluation software automatically. The results have shown that this measurement can provide an estimation of the pearlite colonies size. Light microscopic investigations were found to be unsuitable for determining the pearlite colonies because the optical resolution is insufficient for resolving fine structures such as pearlite lamellae or pearlite colonies.