Serpentine, Mg3Si2O5(OH)4, is a phyllosilicate mineral having several polymorphs, that is, antigorite, chrysotile, lizardite, and polygonal. Because the crystal structure is dependent on thermodynamic environment where serpentine was produced, it has been desired for the polymorphs to be distinguished for geoscientific investigation. While crystal structures are analyzed by X-ray crystallography in many cases, it is difficult to apply the X-ray technique to small and/or non-uniform samples. In the present study, we achieved to unambiguously determine the serpentine's polymorphs with microscopic Raman spectroscopy. Among various Raman-active vibrational modes, we focused on the Si-O-Si symmetric stretching mode and bending vibration of SiO4 units because layer structure of SiO4 tetrahedral sheets (T-sheets) alters with the crystal structure. With the peak wavenumbers determined with subpixel accuracy, the polymorphs were clearly separated in biaxial plots of the peak wavenumbers. As a result, we succeeded in distinguishing lizardite and polygonal serpentine, which were believed to be indistinguishable from Raman spectra, for the first time. Because microscopic Raman spectroscopy is nondestructive method that requires neither large single crystals nor any pretreatment of samples, it is applicable to small (<1 mm), fragile, rare, and/or heterogeneous samples. This feature advantages our technique for the investigation on non-uniform and a trace amount of serpentines in terrestrial mantle rocks from seafloor and in meteorites from asteroids carrying complex histories of their formation and aqueous alteration.