The variation of colors in microscopy systems can be quite critical for some users. To address this problem, a study is conducted to analyze how different factors such as size of the sample, intensity of the microscope’s light source and the characteristics of the material like chroma and saturation can affect the color appearance through the eyepiece of the microscope. To study the changes in colors considering these factors, the spectral reflectance of 24 colors of GretagMacbeth Classic ColorChecker® and Mini ColorChecker® which are placed under a Nikon ECLIPSE MA200 microscope®<sup>2</sup> using dark filed and bright field illuminations which result in different intensity levels, is measured using a spectroradiometer®<sup>3</sup> which was placed in front of the eyepiece of the microscope. The results are compared with the original data from N. Ohta<sup>1</sup>. The evaluation is done by observing the shift in colors in the CIE 1931 Chromaticity Diagram and the CIELAB space, also by applying a wide set of color-difference formulas, namely: CIELAB, CMC, BFD, CIE94, CIEDE2000, DIN99d and DIN99b. Furthermore, to emphasize on the color regions in which the highest difference is observed, the authors have obtained the results from another microscope; Olympus SZX10®<sup>4</sup>, which in this case the measurement is done by mounting the spectroradiometer to the camera port of the microscope. The experiment leads to some interesting results, among which is the consistency in the highest difference observed considering different factors or how the change in saturation of the samples of the same hue can affect the results.