Fourier-transform infrared spectroscopy (FTIR) and second derivative spectra were used to analyze and evaluate the different parts of Cordyceps kyushuensis Kob in the present work. The results showed that C. kyushuensis contained proteins, polysaccharides, nucleosides, lipids and other active ingredients, the single-dimensional IR spectra of the various parts were highly similar, the similarity coefficient between the cultured stroma and medium reached up to 0.992 7, and the natural stroma was more different from the parasites, with similarity coefficient of 0.949 9. Second derivative spectrum further enriched and confirmed the feature of corresponding spectrum peaks, proved the existence of active substances such as cordycepin and adenosine, and prompted the presence of α- and β-glycosidic bonds. The diversity and complexity of chemical constituents in different parts of C. kyushuensis were synthetically described by IR spectra, which provided a fast, comprehensive and objective approach to the analysis and evaluation of the imperceptible differences, and the quality control of Cordyceps. This work supplies a theoretical basis for development and utilization of genetic resources C. kyushuensis.