This study aims to identify the microstructural changes of metal by measuring its natural frequency and damping loss factor. For this purpose, six CK35 steel specimens with ferritic-pearlitic structure were prepared and then, four specimens were spheroidized to significantly change microstructures. Subsequently, the modal testing was applied to determine the vibrational characteristics of specimens with ferrite-pearlite and spheroidized structures, which were identical in terms of size and weight. Experimental results indicated that the dispersion of spheroidized carbides in the ferritic matrix could increase natural frequencies and damping loss factors, such that changing the microstructure of specimens increased the natural frequency and damping loss factor of the first vibration mode by 0.4 (from 3732 to 3747 Hz) and 15%, respectively. Finally, a microstructure-based finite element model was developed to interpret experimental results. The representative volume element (RVE) was modeled using image processing and writing scripts in ABAQUS/CAE 2016. The statistical analysis of stresses calculated by finite element analysis confirmed the results obtained by experimental tests. The numerical results showed that changing microstructures led to considerable changes in stress statistical dispersion, although the mean stress value did not change. Ultimately, the approximate formula of stress-damping could explain significant changes in the damping loss factor.